CONNECTION OF ENDONUCLEASE REPAIR MODEL FOR GENOME ENGINEERING

专利摘要:
the present invention relates to artificial molecular complexes comprising at least one site-specific nuclease and directly interacting with it at least one repair model docking domain, said repair model docking domain interacting with at least one sequence of repair model nucleic acid. the artificial complex may additionally comprise at least one interaction domain. artificial molecular complexes are configured to mediate the repair of a target DNA sequence in a prokaryotic or eukaryotic organism with high precision in a targeted manner and therefore can be used for genome engineering in a cell or prokaryotic organism or eukaryotic / o, or editing a viral genome. methods of modifying at least one target DNA sequence in a prokaryotic or eukaryotic cell or in a viral genome are additionally provided, for example, for trait development, or for treating a disease. in addition, a method is provided for making a plant, a plant cell, a plant material, or a derivative, or a progeny thereof comprising or edited by at least one artificial molecular complex.
公开号:BR112019015578A2
申请号:R112019015578-3
申请日:2018-01-30
公开日:2020-03-10
发明作者:Labs Mathias
申请人:KWS SAAT SE & Co. KGaA；
IPC主号:

专利说明:

Invention Patent Specification Report for
CONNECTION OF REPAIR MODEL TO ENDONUCLEASES FOR
GENOME ENGINEERING.
Technical Field [001] The present invention relates to artificial molecular complexes comprising at least one site-specific nuclease and directly interacting with it at least one repair model docking domain, said interacting repair model docking domain with at least one repair model nucleic acid sequence. An artificial complex can additionally comprise at least one interaction domain. Artificial molecular complexes are configured to mediate the repair of a target DNA sequence in a prokaryotic or eukaryotic or viral organism or genome with high precision in a targeted manner and therefore can be used for genome engineering in engineering a cell or prokaryotic or eukaryotic organism or genome with a prokaryotic, eukaryotic, or viral genome in vivo or in vitro. Methods of modifying at least one target DNA sequence in a prokaryotic or eukaryotic cell, or in a viral genome, for example, for feature development, or for treating a disease, are provided. In addition, a method is provided for making a plant, a plant cell, a plant material, or a derivative, or a progeny thereof comprising or edited by at least one artificial molecular complex. Therefore, an artificial molecular complex suitable for any site-specific nuclease is provided, which directs a repair model in close physical proximity to a target DNA sequence to be modified, in order to allow the prompt availability of a repair model in situ. at the site of a double chain break of
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DNA induced, to ensure high efficiency and predictability for a variety of genome engineering approaches.
Background to the Invention [002] The accuracy of genetic editing or genome engineering has evolved as one of the most important areas of genetic engineering allowing for targeted and targeted manipulation at the site of a genome of interest. An indispensable prerequisite for site-directed genome engineering are programmable nucleases, which can be used to break a nucleic acid of interest into a defined position to induce either a double-strand break (DSB) or a or more single chain breaks. Alternatively, the said nucleases can be chimeric or mutated variants, no longer comprising a nuclease function, but instead operating as recognition molecules in combination with another enzyme. These nucleases or variants of them are therefore essential to any approach to genetic editing or genome engineering. In recent years, many suitable nucleases have been developed, especially adapted endonucleases, comprising meganucleases, zinc finger nucleases, TALE nucleases, Argonauta nucleases, derived, for example, from Natronobacterium gregoryi, and CRISPR nucleases, comprising, for example, Cas nucleases , Cpf1, CasX or CasY as part of the CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats, NT: Regularly Grouped Short Palindromic Repeats).
[003] CRISP Rs (Clustered Regularly Interspaced Short Palindromic Repeats) in their natural environment originally evolved in bacteria where the CRISPR system plays the role of an adaptive immune system to defend against viral attack. After
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3/202 exposure to a virus, short segments of viral DNA are integrated within the CRISPR locus. The RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. This RNA, which contains a sequence complementary to the viral genome, mediates the targeting of a CRISPR effector protein to a target sequence in the viral genome. The CRISPR effector protein cleaves and thus interferes with the replication of the viral target. In recent years, the CRISPR system has been successfully adapted for genetic editing or genome engineering also in eukaryotic cells. Animal cell editing and therapeutic applications for humans are currently important in research. The targeted modification of complex animal and plant genomes is still a demanding task.
[004] A CRISPR system in its natural environment describes a molecular complex comprising at least one small, individual non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease as a Cpf1 nuclease (Zetsche et al., Cpf1 Is a Single RNA -Guides Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, pp. 1-13, October 2015) that can produce a specific double-stranded DNA break. Currently, CRISPR systems are categorized into two classes comprising five types of CRISPR systems, the Type II system, for example, using Cas9 as an effector and the Type V system using Cpf1 as the effector molecule (Makarova et al., Nature Rev. Microbial., 2015). In artificial CRISPR systems, a synthetic non-coding RNA and a CRISPR nuclease and / or optionally a modified CRISPR nuclease, modified to act as a nickase or lacking any nuclease function, can be used in combination with at least one synthetic or artificial guide RNA or gRNA combining the function of a crRNA and / or a tracrRNA (Makarova et
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4/202 al., 2015, supra). The CRISPR / Cas-mediated immune response in natural systems requires CRISPR-RNA (crRNA), in which the maturation of this guide RNA, which controls the specific activation of the CRISPR nuclease, varies significantly between the various CRISPR systems which have been characterized up to the moment. First, the invading DNA, also known as a spacer, is integrated between two adjacent repeating regions at the proximal end of the CRISPR locus. Type II CRISPR systems code for a Cas9 nuclease as a key enzyme for the interference step, whose systems contain both a crRNA and a transactivating RNA (tracrRNA) as the guiding motif. These hybridize and form double stranded RNA regions (ds, double-stranded) which are recognized by RNAse III and can be cleaved to form mature crRNAs. These, in turn, associate with the Cas molecule in order to target the nuclease specifically to the target nucleic acid region. The recombinant gRNA molecules can comprise both the variable DNA recognition region as well as the Cas interaction region, and can be designed specifically, independently of the specific target nucleic acid and the desired Cas nuclease. As an additional safety mechanism, PAMs (adjacent protospacer motifs) must be present in the target nucleic acid region; these are DNA sequences which follow directly from the DNA recognized by the Cas9 / RNA complex. The PAM sequence for Streptococcus pyogenes Cas9 has been described as NGG or NAG (standard IUPAC nucleotide code) (Jinek et al, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 2012, 337: 816-821 ). The PAM sequence for Staphylococcus aureus Cas9 is NNGRRT or NNGRR (N). CRISPR / Cas9 variant systems are known
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Additional 5/202. Thus, a Cas9 of Neisseria meningitidis diva following PAM NNNNGATT. A Cas9 of Streptococcus thermophilus cleaves in the PAM sequence NNAGAAW. An additional NNNNRYAC PAM motif for a Campylobacter CRISPR system (WO 2016/021973 A1) has recently been described. For Cpf1 nucleases, the Cpf1-crRNA complex has been reported to effectively cleave the target DNA proceeded by a short T-rich PAM in contrast to the commonly G-rich PAMs recognized by Cas9 systems (Zetsche et al., Supra). Furthermore, using modified CRISPR polypeptides, specific single chain breaks can be obtained. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific double-stranded DNA breaks through double-stranded DNA. In addition, using two gRNAs, the specificity of DNA binding and, therefore, DNA dividing can be optimized.
[005] Currently, for example, Type II systems based on Cas9, or a variant or any chimeric form thereof, such as endonuclease have been modified for genome engineering. Synthetic CRISPR systems consisting of two components, a guide RNA (gRNA) also called a single guide RNA (sgRNA) and a non-specific CRISPR-associated endonuclease can be used to generate knock-out cells or animals by co-expressing a specific gRNA for the gene to be targeted and capable of association with the Cas9 endonuclease. Notably, gRNA is an artificial molecule comprising a domain interacting with Cas or any other CRISPR effector protein or a catalytically active variant or fragment thereof and another domain interacting with the target nucleic acid of interest and thus representing a synthetic crRNA fusion and tracrRNA (single guide RNA (sgRNA) or simply gRNA; Jinek et al., 2012,
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Supra 6/202). The genomic target can be any DNA sequence of ~ 20 nucleotides, as long as the target is present immediately upstream of a PAM. The PAM sequence is extremely important for target binding and the exact sequence is dependent on the Cas9 species and, for example, reads 5 'NGG 3' or 5 'NAG 3' (standard IUPAC nucleotide code) (Jinek et al., 2012, supra) for a Cas9 derived from Streptococcus pyogenes. Using modified Cas nucleases, targeted single-strand breaks can be introduced into a target sequence of interest. The combined use of a similar Cas nickase with different recombinant gRNAs with highly specific DNA double strand breaks can be introduced using a double cut system. The use of one or more gRNAs can further increase overall specificity and reduce off-target effects.
[006] Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold domain and positively charged grooves with exposed surface in Cas9. It is important to note that the gRNA spacer sequence remains free to interact with the target DNA. The Cas9-gRNA complex will bind to any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cut. As soon as the Cas9-gRNA complex binds to a putative DNA target, a seed sequence at the 3 'end of the gRNA targeting sequence begins to pair (ring) with the target DNA. If the seed and target DNA sequences match, the gRNA will continue to pair with the target DNA in a 3 'to 5' direction (relative to the gRNA's polarity).
[007] Recently, CRISPR / Cpf 1 systems handled in addition to CRISPR / Cas9 systems have become increasingly important
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7/202 for targeted genome engineering (see Zetsche et al., Supra and European patent application No. EP 3 009 511 A2). The Type V system together with the Type II system precedes the CRISPR Class 2 systems (Makarova and Koonin Methods. Mol. Biol., 2015, 1311: 47753). The effector protein Cpf1 is a large protein (about 1,300 amino acids) that contains a nuclease domain similar to RuvC homologous to the corresponding Cas9 domain along with a homolog to the characteristic arginine-rich Cas9 cluster. However, Cpf 1 lacks the HNH nuclease domain that is present in all Cas9 proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9 where it contains long insertions including the HNH domain (Chylinski, 2014; Makarova , 2015). Cpf1 effectors have certain differences in relation to Cas9 effectors, namely, no need for additional transactivating crRNAs (tracrRNA) for CRISPR matrix processing, efficient dividing of target DNA by short T-rich PAMs (in contrast to Cas9, where PAM it is followed by a sequence rich in G), and the introduction of double stranded DNA breaks staggered by Cpf1. Very recently, additional new CRISPR-Cas systems based on CasX and CasY have been identified, which, due to the relatively small size of the effector protein, are of specific interest to many approaches to genetic editing or genome engineering (Burstein et al., New CRISPR-Cas systems from uncultivated microbes, Nature, December 2016). The specificity of CRISPR systems is largely determined by how specific the gRNA targeting sequence is to the genomic target, compared to the rest of the genome.
[008] The Kingdom Plantae comprises species of high heterogeneity and diversity due to the genomic and phenotypic differences of green algae, bryophytes, pteridophytes and
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8/202 terrestrial plants. Plant genomes and their complexity pose a challenge to the high precision of genetic editing or genome engineering. Zea mays [maize] or maize (corn), for example, has the world's largest production of all grain crops, producing 875 million tons in 2012. It has a large genome of about 2.4 gigabases (Gb ) with a haploid chromosome number of 10 (Schnable et al, 2009; Zhang et al, 2009). Triticum aestivum (bread wheat), for example, is hexaploid, with an estimated genome size of ~ 17 Gb. Beta vulgaris ssp. vulgaris (sugar beet) has a genome size ranging from about 470 megabases (Mb) to about 569 Mb. The specific architecture and composition of plant cells and the peculiar development of plants requires a specific adaptation to the engineering tools of genome when intended for use to modify a target sequence within a plant cell. Therefore, the genome engineering tools and the principles associated with it, established for animal systems, particularly mammalian ones, will not necessarily work in a plant cell of interest and there is a need for specific strategies to establish the technology in order to obtain wide application in plants.
[009] Likewise, the genomes of animals, and especially mammals, are complex, for example, comprising 2.7 Gb for the Mus musculus genome or 3.2 Gb for the Homo sapiens genome. Especially, when CRISPR-based genetic editing or genome engineering approaches are intended to be used for precision gene editing or genome engineering of targets within the human genome, there is therefore an urgent need to provide high specificity, a Since any kind of off-target effect can be highly damaging.
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9/202 [0010] Another aspect to be critically considered for genome engineering is the repair mechanism required after dividing a target genomic site of interest, since double-strand breaks (DSBs) or DNA lesions in general are detrimental to the integrity of a genome. Double-strand breaks in genomic material can be caused by ionizing radiation, chemicals, oxidation, enzymes and single-strand breaks during replication and represent a serious form of DNA damage, which can result in gene loss, paralysis of replication. DNA and cell death. Therefore, it is extremely important that the cellular mechanism provides repair mechanisms for double-chain break (DSB). The cells have intrinsic mechanisms to try to repair any damage to double-stranded or single-stranded DNA. DSB repair mechanisms have been divided into two main basic types, non-homologous end junction (NHEJ) and homologous recombination (HR). Homology-based repair mechanisms are usually referred to as homology-directed repair (HOR).
[0011] NHEJ is the dominant nuclear response in animals and plants, which does not require homologous sequences, but is often error-prone and therefore potentially mutagenic (Wyman C., Kanaar R. DNA double-strand break repair: all's well that ends weir, Annu. Rev. Genet. 2006; 40, 363-83). HOR repair requires homology, but HOR pathways that use an intact chromosome to repair the broken, that is, double-chain break repair and synthesis-dependent chain annealing, are highly accurate. In the classic DSB repair pathway, the 3 'ends invade an intact homologous model, then serve as a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (dHJs). At
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10/202 dHJs are branched structures of four chains that form when the invasive chain elongation captures and synthesizes DNA from the second end of the DSB. Individual HJs are resolved by dividing in one of two ways. Synthetic-dependent chain annealing is conservative, and results exclusively in uncrossed events. This means that all newly synthesized sequences are present on the same molecule. Unlike the NHEJ repair pathway, after chain invasion and the formation of the D loop in chain synthesis-dependent annealing, the newly synthesized portion of the invasive chain is displaced from the model and returned to the processed end of the non-invasive chain on the other end of the DSB. The 3 'end of the non-invasive chain is elongated and ligated to fill the gap. There is an additional pathway of HOR, called breakage-induced pathway that is not yet fully characterized. A central feature of this route is the presence of only one invasive end in a DSB that can be used for repair.
[0012] An additional HOR pathway is single chain looping (SSA). SSA is non-conservative and occurs between direct repetitions> 30 bp and results in deletions. In recent years, the microhomology-mediated joint (MMEJ) has been recognized as a distinct type of double-chain break (DSB) repair in eukaryotes. Only very short homology regions (2 to 14 bp) are needed for this route, and typically leave deletions such as SSA. It has also been genetically differentiated from the HR and NHEJ pathways and in mammalian cells acts as a backup for NHEJ (Kwon, T., Huq, E., & Herrin, DL (2010). Microhomology-mediated and nonhomologous repair of a double- strand break in the chloroplast genome of Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 107 (31), 13954-13959). In
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11/202 in short, HR / HOR employs a homologous stretch of DNA on a sister chromatid as a model. It therefore provides high fidelity, however, less efficiency. NHEJ in contrast is highly efficient and a simple and direct route that can rewire the two ends regardless of significant homology, whereas this efficiency is accompanied by the disadvantage that this process is prone to errors and can be associated with insertions or deletions.
[0013] For genetic editing or genome engineering approaches seeking to influence natural repair pathways, they require the physical design of a repair model (RT), which is an important parameter. It may be possible to provide RT either as ssDNA or as partly dsDNA. Current protocols that rely on CRISPR tools for genome editing in combination with a repair model (RT) are based exclusively on the separate provision of nucleic acid RT, either double-stranded or single-stranded, which in turn recognizes the break in the DNA to be repaired solely by base pairing and hybridization. However, the physical and temporal availability of RT at the site where a DNA break is induced cannot be controlled by the methods currently available, since these methods do not provide the precise spatial and temporal provision of RT in the correct configuration, concentration and, therefore, stoichiometric in the compartment, where the repair needs to occur, preferably immediately after the induction of a DNA break directed to specifically control not only the break, but also the repair event.
[0014] Like CRISPR / Cas nucleases, Argonaut endonucleases (Argonauts) are involved in defense against foreign nucleic acids using nucleic acid guides to specify
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12/202 a target sequence, which is then cleaved by the Argonaut protein component. Specifically, an Argonaut can bind to and cleave a target nucleic acid forming a complex with a nucleic acid targeting projected or synthetic nucleic acid, where the dividing of the target nucleic acid can introduce double strand breaks in the target nucleic acid. In addition, like the Cas9 system, the Argonautic nucleic acid guides provide an easy method for programming endonuclease sequence specificity. However, short ssRNA molecules are used as guides by many eukaryotic Argonauts without any recognition limits for secondary structures, such as those present in the Cas9-RNA short guide (sgRNA, gRNA) interaction. Therefore, the abundance of ssRNA in most eukaryotic cells makes the specific targeting of RNA-guided eukaryotic Argonauts a potential challenge. In contrast, some prokaryotic Argonauts are guided by short 5'phosphorylated ssDNA molecules (Swarts, DC et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258-261, 2014; Swarts, DC et al. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA Nucleic Acids Res. 43, 5120-5129 2015), and therefore have inherently less potential for disorientation by host cell-derived nucleic acids due to the shortage of ssDNA molecules short cells in eukaryotic cells. Therefore, DNA-guided Argonauts endonucleases have potential for application in eukaryotic genome editing. However, the use of the Natronobacterium gregoryi Argonauta (NgAgo) system in plants has not been previously demonstrated.
[0015] In the literature, it has been documented that homologous recombination between two sequences occurs more frequently if the sequences are in close proximity within the nucleus, when
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13/202 rather than with a significant amount of separation. For example, the Arabidopsis analysis of the genetic editing rate obtained between donor molecules located chromosomally and targets was greater in both cases where the donor existed on the same chromosome as the target than in other cases where the two loci were located on chromosomes distinct (Fauser et al., 2012). However, these findings have never been explored in a rational way to optimize approaches to genetic editing or genome engineering based on site-specific endonucleases in eukaryotic cells.
[0016] European patent application No. EP 2 958 996 A1 seeks to overcome the specific DSB repair problem by providing an inhibitor of NHEJ mechanisms in the cell to increase nuclease-mediated genetic disruption (for example, ZFN or TALEN) or by a nuclease system (for example, CRISPR / Cas). Inhibiting the critical enzymatic activities of these NHEJ DNA repair pathways, using small molecule inhibitors of the DNA-dependent protein kinase subunit (DNA-PKcs) and / or Poly- (ADP-ribose) polymerase 1/2 (PARP1 / 2), the level of genetic disruption by nucleases is increased, forcing cells to resort to more error-prone repair pathways than classical NHEJ, such as alternating microhomology-mediated union and / or NHEJ. Therefore, an additional chemical is added in the course of genome editing, which, however, can be disadvantageous for various types of cells and assays. This can also affect the genome integrity of the treated cells and / or the regenerative potential.
[0017] Ma et al. (2016, JCB, 214 (5): 529, CRISPR-Cas9 nuclear dynamics and target recognition in living cells) used an sgRNA
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14/202 modified at the 3 'end that made it possible to bind to the aptamer base of a fluorescent reporter to study Cas9 dynamics and sgRNA dynamics for a telomeric target. Noteworthy, the modification within the tracrRNA sequence had no effect on targeting. Only subsequent truncation of the tracrRNA sequence led to destabilized sgRNA regardless of the modification of aptamers.
[0018] Therefore, there is an ongoing need to provide suitable CRISPR tools, particularly tools optimized for precision plant editing, especially main crop plants, which combine high-precision genome divination, for example, providing optimized gRNAs to the target site in a cell of interest and simultaneously providing the possibility to mediate highly precise and accurate homology-directed repair (HOR) and, therefore, targeted repair of a double-strand break, which is imperative to control an intervention of genetic editing or genome engineering.
[0019] Therefore, it is an objective to present new strategies to provide repair models for the accuracy of genome editing, especially convenient for eukaryotic cells, including yeast, animal and plant cells, but also being suitable for prokaryotic cells, for example, for metabolic engineering and various other purposes, or for the modification of viral genomes, for example, to attenuate a virus, or to reduce the virulence of a virus. Despite the tremendous advances in genome editing in biotechnology, for example, for therapeutic approaches, gene therapy or genome engineering of plants or microbes for the development of targeted characteristics, there are still important problems and concerns regarding specificity
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15/202 of a genome modification aimed at being introduced or with respect to off-target effects. This problem, among others, is associated with the degree of precision that can be obtained when inducing a break and the associated repair of a target genomic acid of interest.
[0020] As any type of genetic editing or genome engineering approach inducing a double strand break (DSB) introduces a potentially harmful DNA break and possibly an undesirable DNA repair mechanism leading to undesirable nucleic acid exchanges, there is a continuous need to develop more efficient methods and tools to perform highly accurate and controlled genetic editing or genome engineering, which also implies the use of targeted DNA repair models (RTs).
[0021] Another problem often associated with the provision of successful genome engineering without mediating off-target effects is the physical availability of a repair model at the DSB site just at the time the break is made and therefore needs to be addressed. repaired. Generally, the desired editing event is disadvantaged by repair through the junction of non-homologous ends (NHEJ) or by recombination with an endogenous homologous sequence as detailed above. Depending on the target organism to be modified, this requires a combined strategy for introducing a genetic editing or genome engineering tool together with a repair model of interest so that all tools can, with the appropriate timing, achieve the compartment within a cell comprising the genome, that is, preferably the nucleus, or any other compartment carrying the genome, such as mitochondria. One method of partially overcoming this limitation is by
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16/202 amplification of the repair model and thereby increasing the abundance of the model in the nucleus and presumably making it more available for use for repairing the DSB using a geminivirus vector (see, for example, Mach, Plant Cell. 2014, doi: 10.1105 / tpc.114.122606; and Baltes et al., Plant Cell. 2014, doi: 10.1105 / tpc.113.119792). However, the repair model is released as a separate physical entity and, therefore, there is no control mechanism determining that the repair model will actually be present where DNA repair is needed at exactly the point in time, when a DSB is introduced by an endonuclease.
[0022] With respect to CRISPR applications, there is a frequent suggestion to use free ssDNA nucleotides as repair models or repair models carried by plasmid, although no strategy is revealed or suggested, which would ensure that the repair model were actually brought into physical contact with the double chain break (DSB) to be repaired in situ when a DSB is generated.
[0023] The interactions of biotin-streptavidin and biotin-avidin are among the most stable in nature, with a Kd dissociation constant of 10 ¹⁵ Μ. The association is based on a homotetrameric structure between the avidin or streptavidin protein (-16.5 and 13.2 kDa per subunit, respectively) and the universally present vitamin, but of low abundance. Homotetrameric streptavidin or avidin complexes form spontaneously and are capable of binding to four biotin molecules with low dissociation constants. In at least two attempts, spontaneous tetramerization can be overcome with a decrease in binding affinity (Laitinen et al. 2003, Rational Design of an Active Avidin Monomer. Journal of Biological Chemistry
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278 (6): 4010-4014; Mann et al. 2016, Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin. TECHNOLOGY 4 (3): 1-7). Likewise, it has been shown that biotinylation of a nuclease is possible by including a biotinylation signal in the sequence (Kay et al. 2009, High-throughput Biotinylation of Proteins. Methods in molecular biology (Clifton, NJ) 498: 185-196) . BirA is a possible biotinylation enzyme for expression of bacterial proteins, but biotinylation also occurs in larger plants (Tissot et al. 1996, Protein biotinylation in higher plants: characterization of biotin holocarboxylase synthetase activity from pea (Pisum sativum) leaves, Biochemical Journal 314 (Pt 2): 391-395).
[0024] Single chain variable fragments (scFvs) represent fusion proteins from the heavy (Vh) and light (Vl) variable regions of immunoglobulins, connected with a short peptide linker of ten to about 25 amino acids and are known as versatile high affinity binding molecules. Divalent (or bivalent) single chain variable fragments (di-scFvs, bi-scFvs) can be manipulated by linking two scFvs. This can be accomplished by producing a single peptide chain with two Vh and two Vl regions, producing tandem scFvs (Kufer et al., 2004, Trends in Biotechnology, 22 (5), 238-244; Xiong et al., 2006, Protein Engineering Design and Selection, 19 (8), 359-367).
[0025] However, so far, these discoveries about the capabilities of biotinylated molecules and their cognate binding partners, or about other high affinity molecular binding pairs, such as, for example, single chain antibodies or variable fragments and their Cognate partners have not yet been explored for targeted genome engineering using site-specific nucleases and a repair model.
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18/202 [0026] At this point, the peculiar differences in the release of genetic editing tools or genome engineering and / or repair model as needed for different target cells becomes evident. In this regard, plant cells have some distinct characteristics, including cell walls, making genetic editing or genome engineering in plant cells a completely different task from genetic editing or genome engineering as established for animal / mammalian cells, once that the release of genome editing and / or repair tools is mediated by different methods of transformation, transfection and / or transduction than for other eukaryotic cells. However, these peculiarities need to be taken into account in order to carry out highly accurate plant genome editing. Therefore, it was an object of the present invention to overcome the marked need by providing new tools and methods suitable for high precision genome editing in eukaryotic cells, including plant cells, particularly in the field of genome editing mediated by CRISPR and Argonauta to overcome the existing limitation in the field of genetic editing with reference to the physical availability of the repair model at the site and at the time that the double strand break (DSB) is repaired and, therefore, the competition for DNA repair mechanisms through the end junction pathway is not homologous (NHEJ) or through recombination with homologous (endogenous) sequence (HR / HOR). It was another object of the present invention to provide a simplified site-directed nuclease toolkit for any site-specific nuclease and not being restricted to nucleic acid-guided CRISPR or Argonaut nucleases, which can be used for site-directed genome editing in eukaryotic cells or prokaryotic or any prokaryotic, eukaryotic or viral genome providing a
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19/202 molecule or molecular complex which unifies DNA recognition properties, divage and repair model and simultaneously can be easily released to the target site, that is, a prokaryotic cell, a eukaryotic cell or viral genome, particularly the genome of an animal cell, particularly a mammalian cell, or a plant cell, since the degree of precision to be achieved when editing the genome of animal or plant cells still needs to be improved in order to comply with regulatory requirements necessarily elevated as defined by medical and food administration authorities. The risk for off-target integrations of artificial molecular complexes as disclosed here, in this patent application, is less than for an ss- or ds-DNA repair model introduced as free molecules into the cell. In addition, it was an object to provide a release tool that is specifically optimized to transfer a specific genome editing construct to plants with the aid of a specific release method for plants. In addition, it was an object to provide an approach which can be based on transient editing activity using transiently provided RNA and site-specific nucleases, if desired, due to the sensitivity in certain jurisdictions to any form of genetic modification that integrates foreign DNA as an intermediary in the production process. Finally, it was an object of the present invention to provide a method of genetic editing or genome engineering, which is superior to recent methods in that it saves time relative to experimenting with new targets, since it does not require cloning and pretesting laborious.
Summary of the Invention [0027] The objects identified above were obtained according to
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20/202 with the present invention solving the problem of availability of the repair model by releasing the repair model to the DSB site directly taking advantage of this as a load for the nuclease complex, considering that the spectrum of nucleases suitable for this approach has been dramatically increased by providing artificial molecular complexes, which are based on any site-specific nuclease (SSN) of interest. The targeting of the repair model for the double chain break at the time the break is made in situ providing at least one repair model docking domain (RTDD) along with at least one SSN, in which the docking domain of repair model is configured to interact directly with at least one repair model (RT) nucleic acid sequence increases the local availability of the repair model (RT) for exploration in break repair. In this way, the artificial molecular complexes according to the present invention not only help to provide customized repair models, but in addition they can help to increase the frequency and / or the specificity of genetic editing events. Therefore, this idea combines the functionality of site-specific nuclease and repair models within a single molecular complex for simultaneous genome dividing and targeted repair combined with specific release tools and methods for releasing one or more genome editing tools and / or the repair model within a compartment of interest within a target cell. Therefore, this system allows for greater specificity and thus the reduction of off-target effects of the present editing approaches, which is necessary to minimize off-target divination in genomes of large animals, particularly mammals, or sometimes even plant genomes. complex.
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21/202 [0028] Specifically, the above objects were realized providing, in a first aspect, an artificial molecular complex, comprising (a) at least one site-specific nuclease (SSN) or a catalytically active fragment thereof, or a sequence nucleic acid encoding it, and interacting directly with the same (b) at least one repair model docking domain (RTDD), or a nucleic acid sequence encoding the same, where the repair model docking domain it is configured to interact directly with at least one repair model (RT) nucleic acid sequence; (c) optionally comprising at least one interaction domain (IA), or a nucleic acid sequence encoding the same, wherein the at least one interaction domain is interacting directly with at least one site-specific nuclease or fragment catalytically active, and in which at least one interaction domain is configured to provide at least one of the features selected from the group consisting of (i) interaction with at least one repair model docking domain; and / or (ii) interaction with at least one repair model nucleic acid sequence; and / or (iii) sequence-specific interaction with genomic DNA; wherein the at least one repair model nucleic acid sequence comprises at least a portion being complementary to at least one genomic complementarity sequence, and where at least one repair model nucleic acid sequence is configured to mediate repair of a target DNA sequence.
[0029] In an embodiment according to the various aspects of the present invention, an artificial molecular complex is provided, in which the site-specific nuclease, or the nucleic acid sequence encoding it, is selected from at least one among one
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22/202 CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, a restriction endonuclease, including a class IIS restriction endonuclease, including Fokl or a variant thereof, or two sitiospecific cut endonucleases, or a variant or a catalytically active fragment thereof.
[0030] In another embodiment, an artificial molecular complex is provided, in which at least one repair model docking domain, or the nucleic acid sequence encoding it, is selected from at least one among biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with at least one template nucleic acid sequence repair, a streptavidin, or a variant thereof, preferably a monomeric streptavidin, an avidin, or a variant thereof, an affinity marker, preferably a streptavidin marker, an antibody, a single chain variable fragment (scFv), a single domain antibody (nanobody), an anticalin, an Agrobacterium VirD2 protein or a domain thereof, a Picornavirus VPg, a topoisomerase or a domain thereof, a PhiX174 phage A protein, a PhiX A * protein, a VirE2 protein or a domain thereof, or digoxigenin. Another general knowledge system for interaction is the SNAP marker, for example, fused to a dCas9 as offered by New England Biolabs Inc. (www.neb.com). The SNAP marker is capable of binding to a series of fluorophores, biotin, and other conjugates. The main purpose is to allow visualization, but it would be useful for tethering the repair model, too.
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[0031] In yet another embodiment of the first aspect above, according to the present invention, an artificial molecular complex is provided, wherein the at least one interaction domain, or the nucleic acid sequence encoding the same, is provided. selected from at least one of a DNA binding domain, a streptavidin, or a variant thereof, preferably a monomeric streptavidin, avidin, or a variant thereof, an affinity marker, a biotinylation signal, an acceptor site biotin, a streptavidin marker, an antibody, a single chain variable fragment (scFv), a single domain antibody (nanobody), anticalin, biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with at least one acid sequence repair model nucleic acid, an Agrobacterium VirD2 protein or a domain thereof, a Picornavirus VPg, a topoisomerase or a domain thereof, a PhiX174 phage protein, a PhiX A * protein, a VirE2 protein or a domain of the same, or digoxigenin.
[0032] In yet a further embodiment, an artificial molecular complex is provided, wherein at least one site-specific nuclease and / or at least one repair model nucleic acid sequence and / or at least one domain of The interaction comprises at least one nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence or a chloroplast localization sequence.
[0033] In another embodiment according to the various aspects of the present invention an artificial molecular complex is provided,
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24/202 wherein the at least one repair template nucleic acid sequence comprises at least one end portion, preferably the 3 'end, where this end portion does not interact with any other component of the artificial molecular complex and it is, therefore, configured to hybridize to at least one genomic complementarity sequence in order to mediate repair of the target DNA sequence, and / or in which at least one repair template nucleic acid sequence is provided as a plasmid.
[0034] In yet another embodiment, an artificial molecular complex is provided, in which at least one site-specific nuclease or the catalytically active fragment thereof, or the sequence encoding it, is selected from among a CRISPR nuclease, preferably from one Cas nuclease or a Cpf1, or a Fokl nuclease, or a catalytically active fragment thereof, and the at least one interaction domain, or the sequence encoding the same, is selected from a single chain variable fragment or a monomeric streptavidin.
[0035] In addition, in another embodiment, an artificial molecular complex is provided, in which the complex comprises at least one guide nucleic acid sequence representing at least one repair model docking domain, in which each of the at least at least one guide nucleic acid sequence comprises (i) a first portion of the sequence that is complementary to a target recognition DNA sequence, and (ii) a second portion of the sequence, wherein the second portion of the sequence is configured to interact with at least one site-specific nuclease, and (iii) where at least one guide nucleic acid sequence is physically associated with at least one repair model nucleic acid sequence and therefore forms
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25/202 a hybrid nucleic acid sequence comprising or consisting of at least one RNA or DNA and at least one additional DNA nucleic acid sequence, and (iv) optionally comprising a linker region between at least one nucleic acid sequence guide and at least one repair template nucleic acid sequence, preferably wherein the repair template nucleic acid sequence is associated with the guide nucleic acid sequence at the 3 'end of the guide nucleic acid sequence, and / or where the repair model nucleic acid sequence is associated with the 5 'end of the guide nucleic acid sequence, and / or where the repair model nucleic acid sequence is located within the guide nucleic acid sequence .
[0036] In another embodiment, an artificial molecular complex is provided, wherein the at least one nucleic acid sequence of the repair model and / or at least one guide nucleic acid sequence comprises a nucleotide sequence selected from a sequence of naturally occurring or non-naturally occurring nucleotides, including a synthetic nucleotide sequence, optionally comprising backbone and / or base modifications, wherein the guide nucleic acid sequence comprises a single or partially stranded RNA or DNA nucleotide sequence single-stranded, and wherein the at least one repair template nucleic acid sequence comprises a single-stranded or a double-stranded DNA nucleotide sequence.
[0037] In a further embodiment according to the various aspects of the present invention, an artificial molecular complex is provided, in which at least one site-specific nuclease, or the sequence encoding it, and at least one
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26/202 interaction domain, or the sequence encoding the same, and / or the at least one repair model docking domain, or the sequence encoding the same, are connected by at least one linking domain.
[0038] In one of the modalities provided, at least one site-specific nuclease or the catalytically active fragment thereof, or the sequence encoding it, is independently selected from the group consisting of a Cas polypeptide from Streptococcus spp., including Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria spp., including Neisseria meningitides, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Mycoplasma, Bacteroacid, Flaccobacteria, Flaccobacteria, , Nitratifractor, Mycoplasma, Campylobacter, Candidatus Micrarchaeum acidiphilum ARMAN-1, Parcubacteria (GenBank: APG80656.1), Sulfolobus spp., Including Sulfolobus islandicus HVE10 / 4 (GenBank: ADX81770.1) or REY15A (GenBank: ADX157) and Candidatus Parvarchaeum acidiphilum ARMAN-4, a Cpf 1 polypeptide from an archaea or a bacterium, including a polypeptide deo Cpf1 of Acidaminococcus spp., including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., Including Lachnospiraceae bacterium ND2006, Lachnospiraceae bacterium MC2017, Lachnospiraceae bacterium MA2020, Butyrivibrio proteoclasticus, Candidatus spp.
GW2011_GWC2_44_17, Smithella sp. SCADC, Smithella sp. SC_K08D17, Francisella spp., Including Francisella novicida U112, Eubacterium eligens, Prevotella spp., Or Porphyromonas spp., Or an Argonauta nuclease of Natronobacterium gregoryi (GenBank:
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27/202
AFZ73749.1), Microcystis aeruginosa (Reference Sequence NCBI:
WP_012265209.1 or NCBI Reference String:
WP_002747795.1 or NCBI Reference String:
WP_012265209.1), Halogeometricum pallidum (GenBank: ELZ29017.1), Natrialaba asiatica (Reference Sequence NCBI: WP_006111085.1), Natronorubrum tibetense (Reference Sequence NCBI: WP_006090832.1), Natrinema pellirubrum18 .1), or Synechococcus spp. (NCBI Reference Sequence: WP_011378069.1) or variants and / or functional fragments and / or combinations thereof, including nickases, or nucleases lacking endonucleolytic activity.
[0039] In a second aspect according to the present invention there is provided an artificial molecular complex according to any of the preceding modalities for use in a method of treating a disease, wherein the disease is characterized by at least one genomic mutation and the artificial molecular complex is configured to target and repair at least one genomic mutation. Therefore, a method of treating a disease using the artificial molecular complex according to any of the preceding claims is provided, wherein the disease is characterized by at least one genomic mutation and the artificial molecular complex is configured to target and repair at least a genomic mutation.
[0040] In a further aspect, a plant, a plant cell, a plant material, or a derivative, or a progeny thereof is provided comprising or edited by at least one artificial molecular complex according to any of the aspects and / or previous modalities.
[0041] In yet an additional aspect a method is provided
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Modification of at least one target DNA sequence comprising the following steps: (i) providing at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising at least one genomic complementarity sequence and at least at least one target DNA sequence in a genomic region of interest; (ii) providing at least one artificial molecular complex as defined in any of the foregoing aspects and / or modalities; (iii) contacting at least one artificial molecular complex with at least one target DNA sequence under conditions suitable to effect (a) interaction of at least one site-specific nuclease with at least one target DNA sequence; and (b) complementary base pairing of at least one repair model nucleic acid sequence with at least one genomic complementarity sequence to perform recognition of at least one complementarity sequence and induction of at least one DNA break by at least at least one site-specific nuclease, wherein the at least one repair model nucleic acid sequence directs homologous repair at the site of at least one target DNA sequence; and (iv) obtaining at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising a modification in at least one target DNA sequence.
[0042] In one embodiment of the above, a method of modifying at least one target DNA sequence is provided, wherein at least one repair model nucleic acid sequence and / or at least one docking domain of repair model of the artificial molecular complex is / are provided for at least one cell and / or prokaryotic, eukaryotic / o, or viral genome independently of at least one site-specific nuclease of at least one complex
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29/202 molecular and the at least one artificial molecular complex is assembled, or partially assembled, within the at least one prokaryotic or eukaryotic or viral genome and / or cell.
[0043] In a further embodiment of the above aspect, a method of modifying at least one target DNA sequence is provided, wherein the at least one artificial molecular complex is an artificial molecular complex assembled ex vivo.
[0044] In an additional embodiment of the above aspect, a method of modifying at least one target DNA sequence is provided, wherein the at least one eukaryotic cell is a plant cell, preferably a plant cell of a plant selected from among the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secalee cereale, Trical Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., Including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosifacumum, Nicotiana, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidop sandy sis, Arabidopsis lyrata, Arabidopsis thaliana, Himalayan crucihimalaya, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Brassica rapa, Brassica rapa, Brassica rapa vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus
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30/202 scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp.,
Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa,
Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the plants mentioned above.
[0045] In an additional modality, a method of modifying at least one target DNA sequence is provided, in which the modification of at least one target DNA sequence causes the edition of a characteristic selected from the group consisting of increased production, tolerance to abiotic stress, including water stress (due to drought), osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, saline stress or flooding, biotic stress tolerance including insect tolerance, bacteria tolerance, tolerance to viruses, tolerance to fungi or tolerance to nematodes, resistance to herbicides, including glyphosate, glufosinate, acetolactate synthase (ALS) inhibitors, and Dicamba, resistance to lodging (lodging), flowering time, resistance to breaking, seed color , endosperm composition, nutritional content, or metabolic engineering, including genome editing to allow take a molecular pharming approach in at least one plant cell.
[0046] A method of modifying at least one target DNA sequence is additionally provided further comprising the following step: (v) identification and / or selection of at least one genome and / or prokaryotic / a, eukaryotic / a cell, or viral comprising modification in at least one target DNA sequence.
[0047] In yet another aspect, a method is provided for manufacturing a plant or plant cell comprising the following steps: (i) carrying out a method according to any
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31/202 one of the aspects and / or modalities above, in which at least one eukaryotic cell is a plant cell; (ii) obtaining at least one plant or one progeny from it from at least one plant cell from step (i); (iii) optionally: determining the modification in at least one target DNA sequence in at least one cell of at least one plant or a progeny thereof.
[0048] In one embodiment, a method is provided for manufacturing a plant or plant cell, in which at least one plant or plant cell is selected from a monocot plant or a dicot plant, preferably, in which the plant is selected among the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secalee , Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., Including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana lycidiana, Nicotiana tomentosifan , Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Ara sandy bidopsis, Arabidopsis lyrata, Arabidopsis thaliana, Himalayan Crucihimalaya, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapativa, Brassica rapativ, Brassica rapativ, Brassica rapa vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus
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32/202 scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp.,
Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa,
Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the plants mentioned above.
[0049] In an additional aspect, the use of at least one artificial molecular complex according to any of the above aspects and / or modalities for genome engineering in a prokaryotic, eukaryotic, or viral cell, genome or organism is provided. preferentially in a plant cell or organism.
[0050] Additional aspects and modalities of the present invention can be derived from the subsequent detailed description, the drawings, the sequence listing as well as the attached set of claims.
Brief Description of the Drawings [0051] Figures 1A to D (Figure 1 A to D) show non-limiting examples of possible configurations and different modes of association for different RNA-DNA or DNA-DNA hybrid nucleic acid sequences, the portion of guide nucleic acid representing the at least one repair model docking domain (RTDD) and / or the at least one interaction domain (IA) according to the present invention. (A) Non-covalent Watson-Crick base pairing of a single chain repair (RT) model (ssDNA) to a guide nucleic acid molecule containing the sequence functioning as a sgRNA or tracrRNA or as a gDNA. (B) Covalent association of a single-stranded RT (ssDNA) with the guide nucleic acid molecule. This form can be manufactured by sequential synthesis of the RTDD guide nucleic acid molecule and RT portions as a single molecule, or by linking separate portions to form a single molecule. (Ç)
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Non-covalent association of a double-stranded RT (dsDNA) with the guide nucleic acid molecule. (D) Covalent association of a double-stranded RT (dsDNA) with the guide nucleic acid molecule.
[0052] Figures 2A to C (Figure 2 A to C) show non-limiting examples of possible locations in which the RT can be attached to or associated with a guide nucleic acid molecule such as at least one RTDD and / or the hair least one AI according to the present invention. (A) Covalent or non-covalent association of the single-stranded or double-stranded RT to the 3 'end of the guide nucleic acid molecule. (B) Covalent or non-covalent association of the single-stranded or double-stranded RT to the 5 'end of the guide nucleic acid molecule. (D) Covalent or non-covalent association of the single-stranded or double-stranded RT to the guide nucleic acid molecule. The repair model (RT) portion is shown in white on this and all subsequent Figures.
[0053] Figures 3A to E (Figure 3 A to E) show a non-limiting example for the phased introduction of an edition within a genomic sequence of interest with the site specific nuclease nuclease complex (SSN) revealed here, in this patent application, using a modality of covalent association of RT with the 3 'end of the guide nucleic acid molecule as an example. (A Schematic diagram of the guide nucleic acid molecule in complex with an SSN, for example, NgAgo, Cas, including Cas9, CasX or CasY, or Cpf1. (B) Schematic diagram of the complex linked to the target DNA (genomic DNA (gDNA) ) and indication of the cut sites (black triangles). (C) Schematic diagram of the cleaved target DNA. (D) Schematic diagram of the cleaved target DNA released by SSN and interacting with the repair model (RT) by Watson base pairing. -Crick complementary. (E) Schematic diagram of the repaired target site (gDNA) including editions
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34/202 copied from RT during homologous recombination. The repair model (RT) portion is shown in white in all Figures.
[0054] Figures 4A to C (Figure 4 A to C) show a non-limiting example for the scheme of a nucleic acid-guided endonuclease fusion protein like SSN and a protein or protein domain as an interaction domain (IA ) with the ability to connect directly or indirectly to a repair model (RT). (A) Schematic diagram of the fusion protein referred to as a complex with the target DNA. (B) Schematic diagram of the complex after the double chain break has been introduced. The nucleic acid-guided endonuclease stands out from the target DNA. The fused nucleic acid repair model forms a complex with the target region in a homology-based manner. (C) Schematic diagram of the target DNA after homology-directed repair. Noteworthy, the approach presented uses more than one RTDD to add more precision to the genome engineering complex.
[0055] Figure 5 shows on the left panel a purified nuclease (in this case a CRISPR nuclease) that was fused with an RTDD1 and expressed in E. coli. It was passed over a denaturing SDS gel, with a continuous gradient (4 to 10%) and shows the quantity and purity of the protein. The protein was stained in this gel. The panel on the right shows the mooring. This is a 4% non-denaturing acrylamide gel (Blue Native PAGE) and here the DNA is stained using Red Gel. The FAM-labeled repair model (with RTDD2) was either incubated in the nuclease buffer without or with the nuclease- RTDD1 shown on the left. If the protein was present, tying occurred as seen by DNA being detected at a higher molecular weight level (arrow).
[0056] Figure 6 shows in line 1 a part of the sequence
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35/202 wild from the target site (full length sequence represents SEQ ID NO: 47), in line 2 and line 3 examples for occurrence of INDEL (full length sequences represent SEQ ID NO: 48 and 49), in line 4 the correct HDR event (full length sequence represents SEQ ID NO: 50) and in line 5 shows the repair model (full length sequence represents SEQ ID NO: 51).
[0057] Figure 7 shows the comparison of the normalized HDR efficiency when the repair model is not (left column) and is tied to the nuclease (right column).
Definitions [0058] It should be noted that, as used herein, in this patent application, the forms of the singular one an and the, include references in the plural unless the context clearly dictates otherwise. For example, reference to a component is also intended to include composition of a plurality of components. References to a composition containing a constituent are intended to include constituents other than the so-called constituent. In other words, the terms one a and o, a do not denote a limitation of the quantity, but preferably denote the presence of at least one of the referred item. Each term is intended to encompass its broadest meaning as understood by those skilled in the art and to include all technical equivalents which operate in a similar manner to accomplish a similar purpose.
[0059] The ranges can be expressed here, in this patent application, as from about or approximately or substantially a particular value and / or up to approximately or approximately or substantially or a particular value. When a similar interval is expressed, other
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36/202 example modalities include from that particular value and / or up to the other particular value. Furthermore, the term about means within an acceptable error range for the particular value as determined by a person with regular knowledge of the technique, which will depend in part on how the value is measured or determined, that is, on the limitations of the measurement system. For example, about can mean within an acceptable standard deviation, according to practice in the art. Alternatively, about can mean a range of up to ± 20%, preferably up to ± 10%, more preferably up to ± 5%, and even more preferably up to ± 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2 times, of a value. Where particular values are described in the application and the claims, unless otherwise stated, the term about is implied and in this context means within an acceptable range of error for the particular value.
[0060] By comprising or containing or including it is indicated that at least the compound, element, particle, or stage of the so-called method is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, stages of the method, even if the other said compounds, materials, particles, steps of the method have the same function as that which is called.
[0061] As used herein, in this patent application, nucleic acid means a polynucleotide and includes a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Thus, the terms
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37/202 polynucleotide, nucleic acid sequence, nucleotide sequence and nucleic acid fragment are used interchangeably to denote a RNA and / or DNA polymer that is single-stranded or double-stranded, optionally containing synthetic nucleotide bases, unnatural, or altered. Nucleotides (generally found in their 5 'monophosphate form) are referred to by their unique letter designation as follows: A for adenosine or deoxyadenosine (for RNA or DNA, respectively), C for cytosine or deoxycytosine, G for guanosine or deoxyguanosine, U for uridine, T for deoxythymidine, R for purines (A or G), Y for pyrimidines (C or T), K for G or T, H for A or C or T, I for inosine, and N for any nucleotide. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or fragment thereof, but the nucleic acid does not necessarily need to encode a gene. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more analogs (for example, altered backbone, sugar or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, didesoxynucleotides, cordicepin, 7-deaza-GTP, florophores (e.g., rhodamine or sugar-linked flurescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7guanosine, methylated nucleotides, inosine, thiouridine, 20 pseudouridine, dihydrouridine, queuosine, and wyosine. A nucleic acid according to the present invention can be connected by phosphodiester bonds, for example, as it occurs naturally, or by
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38/202 phosphorothioate bonds, or a mixture of both.
[0062] The terms guide RNA, gRNA or single guide RNA or sgRNA are used interchangeably here, in this patent application, and refer to either a synthetic fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA ), or the term refers to a single RNA molecule consisting only of a crRNA and / or a tracrRNA, or the term refers to a gRNA individually comprising a crRNA portion or a tracrRNA portion. The tracr portion and the crRNA portion therefore do not necessarily need to be present on a covalently attached RNA molecule, but can also be comprised of two individual RNA molecules, which can be associated or can be associated by non-covalent or covalent interaction to provide a gRNA according to the present disclosure. The terms gDNA or sgDNA or guide DNA are used interchangeably here, in this patent application, and refer to either a nucleic acid molecule interacting with an Argonaut nuclease. Both gRNAs and gDNAs as disclosed herein, in this patent application, are referred to as nucleic acid guide (s) or guide nucleic acid (s) due to their ability to interact with a specific site nuclease and to assist in targeting said site-specific nuclease to a genomic target site.
[0063] The terms genetic editing, genome editing and genome engineering are used interchangeably here, in this patent application, and refer to strategies and techniques for the targeted and specific modification of any genetic information or genome of a living organism . In this way, the terms comprise genetic editing, but also the editing of regions other than regions encoding genes in a genome. Beyond
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39/202 this includes editing or engineering nuclear information (if present) as well as other genetic information in a cell. Furthermore, the terms genome editing and genome engineering also comprise epigenetic editing or engineering, that is, the targeted modification of, for example, methylation, histone modification or non-coding RNAs possibly causing transmissible modifications in gene expression.
[0064] The terms nucleotide and nucleic acid with reference to the sequence or a molecule are used interchangeably here, in this patent application, and refer to single- or double-stranded DNA or RNA of natural or synthetic origin. The term nucleotide sequence is therefore used for any DNA or RNA sequence regardless of its length, so that the term comprises any sequence of nucleotides comprising at least one nucleotide, but also any type of oligonucleotide or larger polynucleotide. Therefore, the term (s) refer to natural and / or synthetic deoxyribonucleic acids (DNA) and / or ribonucleic acid (RNA) sequences, which may optionally comprise synthetic nucleic acid analogs. A nucleic acid according to the present disclosure can optionally be codon optimized. Codon optimization implies that the use of codons from a DNA or RNA is adapted for the use of a cell or organism of interest in order to improve the rate of transcription of the recombinant nucleic acid referred to in the cell or organism of interest. The person skilled in the art is well aware of the fact that a target nucleic acid can be modified in one position due to codon degeneration, whereas this modification will still lead to the same amino acid sequence in that position after translation, which is obtained by codon optimization to take into account
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40/202 considering the use of a species-specific codon for a target cell or organism. The nucleic acid sequences according to the present requirement can carry specific codon optimization for the following non-limiting list of organisms: Hordeum vulgare, Sorghum bicolor, Secale cereale, Triticale, Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryza minute, Oryza australiensis, Oryza a / ta, Triticum aestivum, Triticum durum, Triticale, Hordeum bulbosum, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Ma / us domestica, Beta vulgaris, Helianthus annuus, Daucus glochidusus, Daucus glochidusus, Daucus glochidiatus Daucus carota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosiformis, Nicotiana benthamiana, Solanum / ycopersicum, Solanum tuberosum, Coffea caneididisis, Arabia, Arabia, Arabia, sandy, Himalayan Crucihimalaya, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicu m, Capsella bursa-pastoris, Olmarabidopsis pumila, Arabis hirsuta, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncacea, Brassica nigra, Raphanus sativus, Eruca vesicaria sativa, Citrus sinensis, Jatropha curcas, Glycine max, Gossypus s. , Mus musculus, Rattus norvegicus or Homo sapiens.
[0065] As used here, in this patent application, non-native or non-naturally occurring or artificial can refer to a nucleic acid or polypeptide sequence, or any other biomolecule such as biotin or fluorescein that is not found in a native nucleic acid or protein. Non-native can refer to affinity markers. Non-native can refer to mergers. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations,
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41/202 insertions and / or deletions. A non-native sequence can display and / or encode an activity (for example, enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that can also be displayed by the nucleic acid sequence and / or polypeptide to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence can be linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and / or polypeptide sequence encoding a nucleic acid and / or chimeric polypeptide. A non-native sequence can refer to a 3 'hybridizing extension sequence.
[0066] As used here, in this patent application, nucleotide can generally refer to a combination of base-sugar-phosphate. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), inosine triphosphate (ITP) and triphosphate triphosphate , dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. The derivatives mentioned may include, by way of example, and not by way of limitation, [aS] dATP, 7deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used here, in this patent application, can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Examples
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42/202 illustrative dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unmarked or marked in a detectable manner by techniques of general knowledge. Marking can also be done with quantum dots. Detectable markers may include, for example, radioactive markers, fluorescent markers, chemiluminescent markers, bioluminescent markers and enzyme markers. Fluorescent nucleotide markers may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-5 dimethoxy-4'5dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrodamine (R6G), N , N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA), 6-carboxy-Xrodamine (ROX), 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS).
[0067] As used herein, in this patent application, fusion can refer to a protein and / or nucleic acid comprising one or more non-native sequences (e.g., portions). A fusion can be at the N-terminal or Cterminal end of the modified protein, or both, or within the molecule as a separate domain. For nucleic acid molecules, the fusion molecule can be attached at the 5 'or 3' end, or at any suitable position in the medium. A fusion can be a transcriptional and / or translational fusion. A merger because it comprises one or more of the same non-native sequences. A fusion because it comprises one or more of different non-native sequences. A fusion can be a chimera. A fusion can comprise a nucleic acid affinity marker. A merger can comprise a bar code. A fusion can comprise a peptide affinity marker. A merger can provide
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43/202 Argonaut subcellular location (for example, a nuclear location signal (NLS) for targeting the nucleus, a mitochondrial location signal for targeting mitochondria, a chloroplast location signal for targeting a chloroplast, a signal retention of endoplasmic reticulum (ER), and the like). A fusion can provide a non-native sequence (for example, affinity marker) that can be used to screen or purify. A fusion can be a small molecule such as biotin or a dye such as alexa fluor dyes, Cyanine dye3, Cyanine dye. Fusion can provide increased or decreased stability. In some embodiments, a fusion may comprise a detectable marker, including a portion that can provide a detectable signal. Suitable detectable markers and / or portions that can provide a detectable signal can include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein or fluorescent reporter; a quantum dot; and the like. A fusion can comprise a member of a FRET pair, or a fluorophore / donor / quantum dot acceptor pair. A fusion can comprise an enzyme. Suitable enzymes may include, but are not limited to, horseradish peroxidase, luciferase, beta-25 galactosidase, and the like. A fusion can comprise a fluorescent protein. Suitable fluorescent proteins may include, but are not limited to, a green fluorescent protein (GFP), (for example, an Aequoria victoria GFP, Anguilla japonica fluorescent proteins, or a mutant or derivative thereof), a red fluorescent protein, a yellow fluorescent protein, a yellowish green fluorescent protein (for example, mNeonGreen derived from a fluorescent protein
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44/202 tetrameric cephalocordate Branchiostoma lanceolatum) any of a variety of fluorescent and colored proteins. A fusion can comprise a nanoparticle. Suitable nanoparticles can include fluorescent or luminescent nanoparticles, and magnetic nanoparticles, or nanodiamantess, optionally attached to a nanoparticle. Any optical or magnetic property or characteristic of the nanoparticle (s) can be detected. A fusion can comprise a helicase, a nuclease (for example, Fokl), an endonuclease, an exonuclease (for example, a 5'-exonuclease and / or 3'-exonuclease), a ligase, a nickase, a nuclease-helicase ( eg Cas3), a DNA methyltransferase (eg Dam), or a DNA demethylase, a histone methyltransferase, a histone demethylase, an acetylase (including by way of example, but not limited to, a histone acetylase), deacetylase (including by way of example, not limitation, a histone deacetylase), a phosphatase, a kinase, a (co-) transcription activator, a (co-) transcription factor, an RNA polymerase subunit, a repressor transcription, a DNA-binding protein, a DNA structuring protein, a long non-coding RNA, a DNA repair protein (for example, a protein involved in the repair of breaks or single-stranded and / or double-stranded , for example, proteins involved in exc repair base ion, nucleotide excision repair, incompatibility repair, NHEJ, HR, microhomology-mediated end junction (MMEJ), and / or alternative non-homologous end junction (ANHEJ), such as, for example, and non-limiting, HR regulators and HR complex assembly signals), a marker protein, a reporter protein, a fluorescent protein, a linker binding protein (for example, mCherry or a heavy metal binding protein) , a peptide
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45/202 signal (for example, Tat signal sequence), a targeting protein or peptide, a subcellular location sequence (for example, a nuclear location sequence, a chloroplast location sequence), and / or a antibody epitope, or any combination thereof.
[0068] The term catalytically active fragment as used herein, in this patent application, with reference to amino acid sequences denotes the nucleus sequence derived from a given model amino acid sequence, or from a nucleic acid sequence encoding it, comprising all or part of the active site of the model sequence with the proviso that the resulting catalytically active fragment still possesses the activity that characterizes the model sequence, for which the active site of the native enzyme or a variant thereof is responsible. The mentioned modifications are suitable to generate less bulky amino acid sequences but having the same activity as the model sequence, making the catalytically active fragment a more versatile or more stable tool, being sterically less demanding.
[0069] A variant of any site-specific nuclease disclosed herein, in this patent application, represents a molecule comprising at least one mutation, deletion or insertion compared to the wild site-specific nuclease to alter the activity of the naturally occurring wild nuclease . A variant may, as a non-limiting example, be a catalytically inactive Cas9 (dCas9), or a site-specific nuclease, which has been modified to function as a nickase.
[0070] The term release construct or release vector as used here, in this patent application, refers to any biological or chemical medium used as a filler for the
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46/202 transport of a nucleic acid, including a hybrid nucleic acid comprising RNA and DNA, and / or an amino acid sequence of interest into a target cell, preferably a eukaryotic cell. The term release construct or vector as used herein, in this patent application, therefore refers to a means of transport to release a genetic construct or a recombinant construct according to the present disclosure within a target cell, tissue, organ or an organism. A vector, therefore, can comprise nucleic acid sequences, optionally comprising sequences such as regulatory sequences or localization sequences for release, either directly or indirectly, within a target cell of interest or within a target plant structure in the desired cell compartment of a plant. A vector can also be used to introduce an amino acid sequence or a ribonucleo-molecular complex into a target cell or target structure. Generally, a vector as used here, in this patent application, can be a plasmid vector. In addition, according to certain preferred embodiments according to the present invention, a direct introduction of a construct or sequence or complex of interest is conducted. The term direct introduction implies that the target cell or target structure containing a desired DNA target sequence to be modified according to the present disclosure is directly transformed or transduced or transfected into the specific target cell of interest, where the material released with the release vector will exert its effect. The term indirect introduction implies that the introduction is carried out within a structure, for example, leaf cells or cells of organs or tissues, which do not represent the actual target cell or structure of interest to be transformed, but these structures serve as a basis for
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47/202 systemic spread and vector transfer, preferably comprising a genetic construct according to the present disclosure for the actual target structure, for example, a meristematic cell or tissue, or a stem cell or tissue. In case the term vector is used in the context of transfection of amino acid sequences and / or nucleic acid sequences, including hybrid nucleic acid sequences, within a target cell the term vector implies agents suitable for transfection of peptides or proteins, such as, for example, ionic lipid mixtures, cell-penetrating peptides (CPPs), or particle bombardment. In the context of the introduction of nucleic acid material, the term vector may not only imply plasmid vectors, but also suitable carrier materials which may serve as a basis for the release of the introduction of nucleic acid and / or amino acid sequences into a target cell of interest, for example, by particle bombardment. The aforementioned vehicle material comprises, inter alia, gold or tungsten particles. Finally, the term vector also implies the use of viral vectors for the introduction of at least one genetic construct according to the present disclosure, such as, for example, modified viruses, for example, derived from the following virus strains: adenoviral vectors or adeno-associated viruses (AAV), lentiviral vectors, the herpes simplex virus (HSV-1), the vaccinia virus, the Sendai virus, the Sindbis virus, the alphaviruses of the Semliki forest, the Epstein-Barr virus (EBV) , corn streak virus (MSV), barley stripe mosaic virus (BSMV), bromine mosaic virus (BMV, accession numbers: RNA 1: X58456; RNA2: X58457; RNA3: X58458), the corn stripe virus (MSpV), the fine streaked corn virus (MYDV), the yellow dwarf corn virus (MYDV), the dwarf corn mosaic virus (MDMV), the family positive chain RNA viruses Benyviridae,
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48/202 for example, the beet necrotic yellow vein virus (accession numbers: RNA 1: NC_003514; RNA2: NC_003515; RNA3: NC_003516; RNA4: NC_003517) or from the Bromoviridae family, for example, viruses of the genus genus Alfalfa mosaic virus (accession numbers: RNA1: NC_001495; RNA2: NC_002024; RNA3: NC_002025) or of the genus Bromovirus, for example, BMV (supra), or of the genus Cucumovirus, for example, the cucumber mosaic virus ( accession numbers: RNA1: NC_002034; RNA2: NC_002035; RNA3: NC_001440), or of the genus Oleavirus, the dsDNA viruses of the Caulimoviridae family, particularly of the Badnavirus or Caulimovirus family, for example, different banana streak viruses (for example, access numbers: NC_007002, NC_015507, NC_006955 or NC_003381) or the cauliflower mosaic virus (accession number: NC_001497), or the viruses of the genus Cavemovirus, Petuvirus, Rosadnavirus, Solendovirus, Soymovirus or Tungrovirus, the RNA viruses of positive family chain ia Closteroviridae, for example, from the genus Ampelovirus, Crinivirus, for example, the lettuce yellow infectious virus (accession numbers: RNA 1: NC_003617; RNA2: NC_003618) or the tomato chlorosis virus (accession numbers: RNA 1: NC_007340; RNA2: NC_007341), Closterovirus, for example, the yellow beet virus (accession number: NC_001598), or Velarivirus, viruses of single-stranded DNA (+/-) from the Geminiviridae family, for example, the viruses of the Becurtovirus family, Begomovirus, for example the golden yellow bean mosaic virus, the curly tobacco shoot virus, the leaf virus wavy mottled tomato, chlorotic mottled tomato virus, dwarf tomato virus, tomato golden mosaic virus, wavy tomato virus, tomato mottled virus, or tomato virus yellow tomato spot, or Geminiviridae of the genus Curtovirus, for example, the virus
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49/202 of the curly top of the beet, or Geminiviridae of the genus Topocuvirus, Turncurtvirus or Mastrevirus, for example, the corn ray virus (above), the yellow dwarf tobacco virus, the dwarf wheat virus, the RNA viruses of positive chain of the family Luteoviridae, for example, of the genus Luteovirus, for example, the yellow dwarf barley virus-RAV (accession number: NC_004750), or of the genus Polerovirus, for example, the potato leafroll virus (accession number: NC_001747), the single-stranded DNA viruses of the Nanoviridae family, comprising the genus Nanovirus or Babuvirus, the double-stranded RNA viruses of the Partiviridae family, comprising among others the Alphapartitivirus, Betapartitivirus or Deltapartitivirus families, viruses of the Pospiviroidae family, viruses of positive-stranded RNA from the Potyviridae family, for example, comprising the genus Brambyvirus, Bymovirus, Ipomovirus, Macluravirus, Poacevirus, for example, the Trit mosaic virus icum (accession number: NC_012799), or Potyviridae of the genus Potyvirus, for example, the beet mosaic virus (accession number: NC_005304), the dwarf corn mosaic virus (accession number: NC_003377), the Y virus potato (accession number: NC_001616), or the Zea mosaic virus (accession number: NC_018833), or Potyviridae of the genus Tritimovirus, for example, the Brome streak mosaic virus (accession number: NC_003501) or wheat streak mosaic virus (accession number: NC_001886), single-stranded RNA viruses of the Pseudoviridae family, for example, of the genus Pseudovirus, or Sirevirus, double-stranded RNA viruses of the Reoviridae family, for example, dwarf rice virus (accession numbers: RNA1: NC_003773; RNA2: NC_003774; RNA3: NC_003772; RNA4: NC_003761; RNAS:
NC_003762; RNA6: NC_003763; RNA7: NC_003760; RNAB:
NC_003764; RNA9: NC_003765; RNA10: NC_003766; RNA11:
NC_003767; RNA 12: NC_003768), the chain RNA viruses
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50/202 positive of the Tombusviridae family, for example, comprising the genus Alphanecrovirus, Aureusvirus, Betanecrovirus, Carmovirus, Dianthovirus, Gallantivirus, Macanavirus, Machlomovirus, Panicovirus, Tombusvirus, Umbravirus or Zeavirus, for example, the necrotic corn virus (necrotic corn number) access code: NC_007729), or the positive chain RNA viruses of the Virgaviridae family, for example, the viruses of the genus Furovirus, Hordeivirus, for example, the barley stripe mosaic virus (accession numbers: RNA1: NC_003469; RNA2 : NC_003481; RNA3: NC_003478), or of the genus Pecluvirus, Pomovirus, Tobamovirus or Tobravirus, for example, the tobacco rattle virus (accession numbers: RNA1: NC_003805; RNA2: NC_003811), as well as the chain RNA viruses negative reaction of the order Mononegavirales, particularly of the family Rhabdoviridae, for example, the striated yellow barley mosaic virus (accession number: KM213865) or the necrotic yellow lettuce virus (number access / specimen: NC_007642 / AJ867584), positive-chain RNA viruses of the order Picornavirales, particularly from the Secoviridae family, for example, from the genus Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus, Torradovirus, or Waikavirus, viruses of positive-stranded RNA of the order Tymovirales, particularly from the Alphaflexiviridae family, for example, the viruses of the genus Allexivirus, Lolavirus, Mandarivirus, or Potexvirus, Tymovirales, particularly from the Betaflexiviridae family, for example, the viruses of the genus Capillovirus, Carlavirus, Citrivirus, Foveavirus, Tepovirus, or Vitivirus, the positive-stranded RNA viruses of the order Tymovirales, particularly the family Tymoviridae, for example, the viruses of the order Maculavirus, Marafivirus, or Tymovirus, and bacterial vectors, such as Agrobacterium spp., such as, for example, Agrobacterium tumefaciens. Finally, the term vector also implies chemical transport agents suitable for the introduction of
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51/202 linear nucleic acid sequences (single-stranded or double-stranded) within a target cell combined with a physical introduction method, including polymeric or lipid-based release constructs.
[0071] Release constructs or suitable vectors therefore comprise biological means for the release of nucleotide sequences within a target cell, including viral vectors, Agrobacterium spp., Or chemical release constructs, including nanoparticles, for example, mesoporous silica nanoparticles (MSNPs), cationic polymers, including approaches based on PEI polymers (polyethyleneimine) or polymers such as DEAE-dextran, or non-covalent surface attachment of PEI to generate cationic surfaces, lipid or polymeric vesicles, or combinations thereof. Lipid or polymeric vesicles can be selected, for example, from lipids, liposomes, lilpid encapsulation systems, nanoparticles, formulations of small particles of nucleic acid-lipids, polymers, and polymersomes.
[0072] The terms genetic construct or recombinant construct are used here, in this patent application, to refer to a construct comprising, among others, plasmids or plasmid vectors, cosmids, yeast artificial chromosomes or bacterial artificial chromosomes (YACs and BACs ), phagemids, bacterial phage vectors, an expression cassette, isolated single- or double-stranded nucleic acid sequences, comprising DNA and RNA sequences, or amino acid sequences, viral vectors, including modified viruses, and a combination or a mixture thereof, for introduction or transformation, transfection or transduction within any prokaryotic or eukaryotic target cell, including a plant, plant cell, tissue, organ or material in accordance with this
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52/202 revelation. A recombinant construct according to the present disclosure can comprise an effector domain, either in the form of a nucleic acid sequence or an amino acid sequence, in which an effector domain represents a molecule, which can have an effect on a cell target and includes a transgene, a single-stranded or double-stranded RNA molecule, including a guide RNA (s) gRNA, a miRNA or a siRNA, or certain amino acid sequences, including, but not limited to, an enzyme or a catalytically active fragment thereof, a binding protein, an antibody, a transcription factor, a nuclease, preferably a specific site nuclease, and the like. In addition, the recombinant construct can comprise regulatory sequences and / or localization sequences. The recombinant construct can be integrated into a vector, including a plasmid vector, and / or it can be present isolated from a vector structure, for example, in the form of a polypeptide sequence or as a single-stranded nucleic acid or double strand connected to non-vector. After its introduction, for example, by transformation, the genetic construct may or may persist extrachromosomally, that is, not integrated within the genome of the target cell, for example, in the form of a double-stranded or single-stranded DNA, an RNA double-stranded or single-stranded or as an amino acid sequence. Alternatively, the genetic construct, or parts thereof, according to the present disclosure can be integrated stably within the genome of a target cell, including the nuclear genome or additional genetic elements of a target cell, including the genome of plastids such as mitochondria or chloroplasts. The term plasmid vector as used in this context refers to a genetic construct obtained originally from a plasmid. A plasmid usually refers to a
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53/202 extrachromosomal element replicating autonomously circular in the form of a double-stranded nucleic acid sequence. In the field of genetic engineering, these plasmids are routinely subjected to targeted modifications by inserting, for example, genes encoding a resistance against an antibiotic or herbicide, a gene encoding a target nucleic acid sequence, a localization sequence, a regulatory sequence , a marker sequence, a marker gene, including an antibiotic marker or a fluorescent marker, and the like. The structural components of the original plasmid, such as the origin of replication, are maintained. According to certain embodiments of the present invention, the localization sequence can comprise a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence or a chloroplast localization sequence. The location sequences referred to are available to the person skilled in the art of plant biotechnology. A variety of plasmid vectors are commercially available for use in different target cells of interest and their modification is known to the person skilled in the respective art.
[0073] The term genetically modified or genetic manipulation or genetically manipulated is used in a broad sense here, in this patent application, and means any modification of a nucleic acid sequence or an amino acid sequence, a target cell, tissue, organ or organism, which is performed by human intervention, either directly or indirectly, in order to influence the endogenous genetic material or the transcriptome or proteome of a target cell, tissue, organ or organism to modify it in an intentional way so that differs from its state as it is found without human intervention, whereas the
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54/202 The term genome editing specifically refers to a targeted manipulation of the genome of a target cell. Human intervention can occur either in vitro or in vivo, or both. Additional modifications can be included, for example, one or more point mutations, for example, for targeted protein engineering or for codon optimization, one or more deletions, and one or more insertions or one or more deletions of at least one molecule of nucleic acid or amino acid (also including homologous recombination), modification of a nucleic acid or sequence of amino acids, or a combination thereof. The terms will also comprise a nucleic acid molecule or an amino acid molecule or a host cell or an organism, including a plant or plant material of the same which is similar to a sequence, an organism or a comparable material as they occur in nature, but which have been built by at least one stage of intentional manipulation.
[0074] A targeted genetic manipulation or targeted or site-directed genetic editing or genome editing as used here, in this patent application, is therefore the result of genetic manipulation, which is carried out in a targeted manner, that is , at least one specific position in a target cell and under the appropriate specific circumstances to obtain a desired effect in at least one cell, preferably a plant cell, to be manipulated.
[0075] The term transgenic as used in accordance with the present disclosure refers to an animal, an animal cell, tissue or organ, a plant, plant cell, tissue, organ or material which comprises a gene or genetic construct, comprising a transgene that has been transferred into the plant, the cell
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55/202 plant, tissue, organ or material by natural means or by means of genetic engineering techniques from another organism. The term transgene comprises a nucleic acid sequence, including DNA or RNA or a combination or mixture thereof. Therefore, the term transgene is not restricted to a sequence commonly identified as a gene, that is, a sequence encoding protein. It can also refer, for example, to a DNA or RNA sequence not encoding protein. Therefore, the term transgenic generally implies that the respective nucleic acid introduced into a cell of interest is not naturally present in the respective target prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a fungal cell, an animal or a cell animal, plant, plant cell, tissue, organ or material. The terms transgene or transgenic as used herein, in this patent application, therefore refer to a nucleic acid sequence or an amino acid sequence that is taken from an organism's genome, or produced synthetically, and which is then introduced into another organism, in a transient or stable mode, by artificial techniques of molecular biology, genetics and the like.
[0076] The term plant or plant cell as used here, in this patent application, refers to a plant organism, a plant organ, differentiated and non-differentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof. Plant cells include, without limitation, for example, seed cells, mature and immature embryos, meristematic tissues, seedlings, callus tissues in different states of differentiation, leaves, flowers, roots, buds, gametophytes, sporophytes, pollen, pollen and microspores, protoplasts, macroalgae and microalgae. The different plant cells can be either haploid,
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56/202 diploids, tetraploids, hexaploids or polyploids.
[0077] Subject, as used here, in this patent application, can mean either a human or a non-human animal. The term includes, but is not limited to, mammals (for example, humans, other primates, pigs, rodents (for example, mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep , and goats). In one embodiment, the subject is a human being.
[0078] Treating, treating and treating, as used here, in this patent application, generally means obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or a symptom of it and / or it can be therapeutic in terms of a partial or complete cure for a disease and / or an adverse effect attributable to the disease. Treatment as used herein, in this patent application, covers any treatment of a disease or symptom in a mammal, and includes: (a) prevention of the occurrence of the disease or symptom in a subject who may be predisposed to acquire the disease or the symptom but has not yet been diagnosed as having it; (b) inhibition of the disease or symptom, that is, interruption of its development; or (c) relieving the disease, that is, causing the disease to regress. The therapeutic agent can be administered before, during or after the onset of the disease or injury. The treatment of existing disease, where treatment stabilizes or reduces the patient's undesirable clinical symptoms, is of particular interest. The referred treatment is desirably performed before complete loss of function in the affected tissues. The subject's therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
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57/202 [0079] A plant material as used here, in this patent application, refers to any material which can be obtained from a plant during any stage of development. The plant material can be obtained either in plant or from an in vitro culture of the plant or tissue from the plant or organ thereof. The term therefore comprises plant cells, tissues and organs as well as developed plant structures as well as sub-cellular components such as nucleic acids, polypeptides and all plant chemical substances or metabolites which can be found within a plant cell or plant compartment and / or which can be produced by the plant, or which can be obtained from an extract of any plant cell, tissue or plant at any stage of development. The term also comprises a derivative of plant material, for example, a protoplast, derived from at least one plant cell comprised of plant material. The term therefore also comprises meristematic cells or meristematic tissue from a plant.
[0080] As used here, in this patent application, the terms mutation and modification are used interchangeably to refer to a deletion, insertion, addition, substitution, editing, chain break, and / or introduction of an adduct in the context nucleic acid manipulation in vivo or in vitro. A deletion is defined as a modification to a nucleic acid sequence in which one or more nucleotides are missing. An insertion or addition is the modification to a nucleic acid sequence which has resulted in the addition of one or more nucleotides. A substitution or editing results from the replacement of one or more nucleotides by a molecule which is a different molecule from the one or more nucleotides substituted. For example, a nucleic acid
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58/202 can be replaced by a different nucleic acid as exemplified by replacing a thymine with a cytosine, adenine, guanine, or uridine. Pyrimidine to pyrimidine (for example, substitutions of nucleotides C to T or T to C) or purine to purine (for example, substitutions of nucleotides G to A or A to G) are called transitions, whereas pyrimidine to purine or purine to pyrimidine (for example, G for T or G for C or A for T or A for C) are called transversions. Alternatively, a nucleic acid can be replaced with a modified nucleic acid as exemplified by replacing a thymine with a thymine glycol. Mutations can result in an incompatibility. The term incompatibility refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different nucleotide sequence or nucleic acid molecule, which does not follow the rules for base pairing. For example, for the partially complementary sequences 5'-AGT-3 'and 5'-AAT-3', one is present G-A incompatibility (one transition).
[0081] The term strand break when made in reference to a double stranded nucleic acid sequence, for example, a genomic sequence as a target DNA sequence, includes a single strand break and / or a double strand break. A single strand break [a nick] refers to an interruption in one of the two strands of the double stranded nucleic acid sequence. This is in contrast to a double strand break which refers to an interruption in both strands of the double stranded nucleic acid sequence. Chain breaks according to the present disclosure can be introduced into a double-stranded nucleic acid sequence by enzymatic incision at a nucleic acid base position of interest using a suitable endonuclease, including a CRISPR endonuclease or a variant of the
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59/202 same, where the variant can be a mutated or truncated version of the wild protein or wild endonuclease, which can still exercise the enzymatic function of the wild protein.
[0082] Complementary or complementarity as used here, in this patent application, describes the relationship between two DNAs, two RNAs, or, with respect to hybrid sequences according to the present invention, between an RNA nucleic acid region and a DNA . Defined by the DNA or RNA nucleobases, two nucleic acid regions can hybridize to each other according to the key and lock model. To this end, the Watson-Crick base matching principles have adenine and thymine / uracil bases as well as guanine and cytosine, respectively, as complementary bases apply. In addition, non-Watson-Crick pairing as well as reverse Watson-Crick, Hoogsteen, reverse Hoogsteen and Wobble pairing are comprised of the complementary term as used here, in this patent application, provided that the respective pairs of bases can build hydrogen bond to each other, that is, two different nucleic acid strands can hybridize to each other based on said complementarity. There is no need for perfect complementarity in the sense of two stretches of sequences aligning 100% to each other over a given extension, since the person skilled in the art is aware of the fact that nucleic acid hybridization is impacted by factors such as the degree and the extent of complementarity between the nucleic acids, the severity of the conditions involved, the Tm of the hybrid formed, and the proportion of G: C within the nucleic acids, and so on. In addition, steric factors can influence the fact that two sequences, although not 100% complementary to each other, will hybridize. Therefore, two complementary nucleic acid sequences from
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60/202 according to the present invention can have at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology or complementarity with each other and can still hybridize to each other under conditions of medium severity. Medium severity conditions refer to 0.165 to 0.330 M NaCI in a temperature range of 20 to 29Ό below Tm, where Tm is defined as the Tm for a DNA sequence that can be estimated using the commonly used calculation:
[0083] Tm = 81.5 + 16.6 logw ([Na +] / 1.0 + 0.7 [Na +]) + 0.41 (% [G + C]) (500 / n) -PF, in that [0084] Tm = melting temperature in ° C, [Na ⁺ ] = molar concentration of sodium ions,% [G + C] = percentage of G + C bases in the DNA sequence, n = length of the DNA sequence on bases P = temperature correction for% of incompatible base pairs (~ 1 ° C for 1% incompatibility), and F = correction for formamide concentration (= 0.63 ° C for 1% of [formamide]).
[0085] The term transitional introduction as used herein, in this patent application, refers to the transitional introduction of at least one nucleic acid sequence in accordance with the present disclosure, preferably incorporated within a release vector or within a recombinant construct, with or without the help of a release vector, within a target structure, for example, a plant cell, in which at least one sequence of
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61/202 nucleic acid is introduced under suitable reaction conditions so that no integration of at least one nucleic acid sequence takes place within the endogenous nucleic acid material of a target structure, of the genome as a whole, so that the at least one nucleic acid sequence will not be integrated into the target cell's endogenous DNA. Consequently, in the case of transient introduction, the introduced genetic construct will not be transmitted to a progeny of the target structure, for example, a prokaryotic cell, an animal cell or a plant cell. At least one nucleic acid sequence or the products resulting from its transcription or translation are only present temporarily, that is, in a transient way, in a constitutive or inducible form, and therefore can only be active in the target cell to exercise its effect for a limited time. Therefore, at least one nucleic acid sequence introduced by means of transient introduction will not be transmissible to the progeny of a cell. The effect that a nucleic acid sequence introduced in a transient mode can, however, potentially be transmitted to the progeny of the target cell.
[0086] The term stable or stably integrated as used herein, in this patent application, refers to the stable integration of at least one nucleic acid sequence according to the present disclosure, preferably incorporated within a vector of release or within a recombinant construct. Integration can occur either within the nuclear genome of a target cell or into any other extranuclear genomic material within a compartment of a eukaryotic cell of interest, for example, a mitochondria or a plastid from a plant cell. Therefore, at least one stable integrated recombinant construct will be
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62/202 transmissible to the progeny of a target cell modified in this way. Depending on the nature of the genetic construct, all or part of the genetic construct will be integrated in a stable manner, since the genetic construct can comprise several regions of interest comprising a target region to be integrated in a stable manner as well as additional regions, among others, necessary for the transport, release, maintenance, and the correct location of the genetic construct within a plant cell, whose regions, however, cannot be the same integrated, but serve as cargo for the region of interest to be integrated in stable manner as is known to the person skilled in the art. The stable integration of at least one genetic construct according to the present disclosure into at least one hematopoietic or meristematic cell or tissue, consequently will lead to the transmission of the thus modified genomic region of the target structure, that is, a target region of DNA, for the progeny of the modified cell through all stages of development of said at least one hematopoietic or meristematic cell, which may be favorable for approaches, where a targeted genetic modification in and the production of the final cell type resulting from differentiation and the development of at least one hematopoietic or meristematic cell. Therefore, obtaining, for example, a stable integration within at least one meristematic cell of the immature inflorescence of a plant can lead to the stable transmission of the genetic characteristic introduced into the pollen gamete or the egg resulting from the development of at least one cell meristem of immature inflorescence. Stable integration within at least one pluripotent hematopoietic cell or any pluripotent or multipotent cell will likewise lead to stable transmission of the genetic trait
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63/202 introduced.
[0087] The term particle bombardment as used here, in this patent application, also called biolistic transfection or microparticle-mediated gene transfer, refers to a physical release method for the transfer of a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest within a target cell or tissue. The micro- or nanoparticle acts as a projectile and is fired at the target structure of interest under high pressure using a suitable device, often called a gene gun. Transformation through particle bombardment uses a metal microprojectile covered with the gene of interest, which is then fired at target cells using equipment known as a gene gun (Sandford et al. 1987) at high speed enough to penetrate the cell wall of a target tissue, but it does not last long enough to cause cell death. For protoplasts, which have their cell wall entirely removed, the conditions are logically different. The precipitated nucleic acid or genetic construct on the at least one microprojectile is released into the cell after bombardment, and either integrated into the genome or transiently expressed according to the definition given above. The acceleration of microprojectiles is accomplished by high voltage electrical discharge or compressed gas (helium). With respect to the metal particles used, it is mandatory that they be non-toxic, non-reactive, and that they have a smaller diameter than the target cell. The most commonly used are gold or tungsten. There is a lot of publicly available information from the manufacturers and suppliers of gene guns and associated systems regarding their general use.
[0088] The derived or descending term or progeny
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64/202 as used herein, in this patent application, in the context of a prokaryotic cell or a eukaryotic cell, preferably an animal cell and more preferably a plant or plant cell or plant material according to the present disclosure refers to descendants of a cell or similar material which result from natural reproductive propagation including sexual and asexual propagation. It is common knowledge of a person specialized in the technique that the propagation referred to can lead to the introduction of mutations within the genome of an organism resulting from natural phenomena which results in a descendant or progeny, which is genomically different from the organism or parental cell, in the However, it still belongs to the same genus / species and has mostly the same characteristics as the parental recombinant host cell. Said derivatives or descendants or progeny resulting from natural phenomena during reproduction or regeneration are, therefore, included in the term of the present disclosure. In addition, the term derivative may imply, in the context of a substance or molecule rather than referring to a cell or organism, directly or through modification obtained indirectly from another. This may involve a nucleic acid sequence derived from a cell or a plant metabolite obtained from a cell or material. Therefore, these terms do not refer to any derivative, descendant or arbitrary parent, but instead to a derivative, or descendant or parent phylogenetically associated with, that is, based on, a cell or virus of origin or a molecule thereof, considering that this relationship between the child, descendant or parent and the parent is clearly inferable by a person skilled in the art.
[0089] In addition, the terms derived, derived from, or
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65/202 derivative as used here, in this patent application, in the context of a biological sequence (nucleic acid or amino acid) or molecule or complex imply that the respective sequence is based on a reference sequence, for example, from the sequence listing, or a database accession number, or the respective scaffold structure, that is, originating from said sequence, considering that the reference sequence can comprise more sequences, for example, the entire genome or a sequence encoding the complete polyprotein, of a virus, whereas the sequence derived from the native sequence can comprise only an isolated fragment thereof, or a coherent fragment thereof. In this context, it can be said that a cDNA molecule or an RNA is derived from a DNA sequence serving as a molecular model. The person skilled in the art can therefore easily define a sequence derived from a reference sequence, which, by sequence alignment at the DNA or amino acid level, will have a high identity with the respective reference sequence and which will have coherent stretches of DNA / amino acids in common with the respective reference sequence (> 75% query identity for a given length of the aligned molecule as long as the derived sequence is the query and the reference sequence represents the subject during an alignment sequences). The person skilled in the art can therefore clone the respective sequences based on the disclosure provided here, in this patent application, through polymerase chain reactions and the like in a suitable vector system of interest, or use a sequence as a scaffold of vector. The term derived from therefore is not an arbitrary sequence, but a sequence corresponding to a reference sequence from which it is derived, considering that certain
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66/202 differences, for example, certain mutations that occur naturally during the replication of a recombinant construct within a host cell, cannot be excluded and, therefore, are comprised by the term derived from. In addition, several stretches of a parental sequence can be concatenated into a sequence derived from the parental one. The different sections will have high homology (preferably more than 90%) or even 100% homology with the source sequence. The person skilled in the art is well aware of the fact that a sequence of artificial molecular complexes according to the present invention, when provided or partially provided as a nucleic acid sequence, will then be transcribed and optionally translated in vivo and possibly further digested. and / or processed within a host cell (signal peptide dividing, endogenous biotinylation, etc.) so that the term derived from indicates a correlation with the sequence originally used in accordance with the disclosure of the present invention.
[0090] The term target region, target site, target structure, target construct, target nucleic acid or target cell / tissue / organism, or target DNA region as used herein, in this patent application, refers to a target which can be any genomic region within any compartment of a target cell.
[0091] The term regulatory sequence as used here, in this patent application, refers to a nucleic acid sequence or an amino acid sequence, which can direct the transcription and / or translation and / or modification of a nucleic acid sequence of interest.
[0092] The terms protein, amino acid or polypeptide are
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67/202 used interchangeably here, in this patent application, and refer to an amino acid sequence having a catalytic enzymatic function or a structural effect or a functional effect. The term amino acid or sequence of amino acids or amino acid molecule comprises any natural or chemically synthesized protein, peptide, polypeptide and enzyme or a modified protein, peptide, polypeptide and enzyme, wherein the modified term comprises any chemical or enzymatic modification of the protein, peptide , polypeptide and enzyme, including wild-type sequence truncations for a shorter but still active portion.
[0093] In accordance with the present invention, conventional techniques of molecular biology, microbiology, and recombinant DNA can be employed within the knowledge of the art. The referred techniques are fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (here, in this patent application, Sambrook et al., 1989) ; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ. Higgins eds. (1985); Transcription and Translation (BD Hames & SJ Higgins, eds. (1984); Animal Cell Culture (Rl. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); others.
[0094] Whenever the present disclosure refers to the percentage of homology or identity of nucleic acid sequences or amino acid sequences, these values define those obtained using
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68/202 the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program (www.ebi .ac. Uk / T ools / psa / emboss_water / nucleotide.html) for nucleic acid sequences or the EMBOSS Water Pairwise Sequence Alignments (protein) program ( www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences. The tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk/Tools/psa/ and Smith, TF & Waterman , MS Identification of common molecular subsequences Journal of Molecular Biology, 1981 147 (1): 195-197). When conducting an alignment, the default parameters defined by EMBL-EBI are used. These parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap opening penalty = 10 and gap extension penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap opening penalty interval = 10 and interval extension penalty = 0.5. Detailed Description [0095] According to the first aspect of the present invention, an artificial molecular complex is provided, comprising (a) at least one site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding it, and interacting directly with it (b) at least one repair model docking domain (RTDD), or a nucleic acid sequence encoding the same, where the repair model docking domain is configured to interact directly with at least one repair model (RT) nucleic acid sequence; (c) optionally comprising at least one interaction domain (IA), or a nucleic acid sequence encoding the same, wherein the at least one interaction domain
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69/202 is interacting directly with at least one site-specific nuclease or the catalytically active fragment thereof, and in which at least one interaction domain is configured to provide at least one of the features selected among the group consisting of (i) interaction with at least one repair model docking domain; and / or (ii) interaction with at least one repair model nucleic acid sequence; and / or (iii) sequence-specific interaction with genomic DNA; wherein the at least one repair model nucleic acid sequence comprises at least a portion being complementary to at least one genomic complementarity sequence, and where at least one repair model nucleic acid sequence is configured to mediate repair of a target DNA sequence.
[0096] The present invention, therefore, is based on a site-specific nuclease (SSN). This nuclease is characterized by having a nuclease function and a DNA recognition function. The DNA recognition function can be intrinsic to the nuclease in the form of a domain mediating recognition or binding to DNA, or it can be assisted by additional guide molecules, for example, for CRISPR nucleic acid-guided (RNA-guided) or Argonauta nucleases (guided by DNA), but the present invention is not restricted to the use of the nucleic acid-guided nucleases mentioned above and therefore increases the scope of genome engineering targeted to any non-CRISPR or Argonauta site-specific nucleases. Another part of the artificial system according to the present invention is at least one repair model (RT) nucleic acid sequence, since the products and methods of the present disclosure essentially focus on making a RT physically available at a site of a double strand break induced by an SSN. In addition, the present invention is based on
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70/202 a repair model docking domain (RTDD) as part of an optimized molecular system. This RTDD fulfills the function of bringing directly or indirectly the SSN and at least one RT in close contact to allow efficient and targeted genome engineering.
[0097] The RTDD is, therefore, associated in a covalent or non-covalent manner with the RT, that is, it is interacting directly with the RT on a molecular level. Simultaneously, the RTDD is interacting directly with at least one SSN and, therefore, represents the binding molecule or domain between the SSN and the RT. For RTDD, there are several possible configurations. In one mode, RTDD is directly associated with SSN. For example, if the SSN is a CRISPR nuclease, the RTDD can be a gRNA, or if the SSN is an Argonauta nuclease, the RTDD can be a gDNA. In another embodiment, the RTDD can be part of the SSN itself, or it can be part of the RT, the RTDD representing a specific portion of the RT or the SSN. In these modalities, the SSN can comprise a domain as part of its amino acid sequence, which can interact with an aptamer carrying an RT. Therefore, in certain modalities, there is no separate interaction domain, since the site-specific nuclease itself comprises a domain for interaction with an RTDD. The RTDD can therefore be an aptamer associated with the RT nucleic acid sequence, in which the aptamer is recognized and can therefore interact specifically or with the SSN and / or with an additional interaction domain.
[0098] Furthermore, covalent and non-covalent interactions between the components of the artificial molecular complex according to the present invention are envisaged.
[0099] In an additional modality, the molecular complex
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The artificial 71/202 comprises an additional interaction domain (AI). In this configuration, RTDD can be associated with the additional interaction domain for certain modalities. The interaction domain is interacting directly, that is, physically associated, either covalently as a fusion molecule or non-covalently, with SSN and provides additional functionality for the molecular complex. The interaction domain can be a protein domain comprising DNA recognition / binding functions, that is, it can be a domain which is capable of interacting with a target site of genomic DNA in a site-specific manner, or the domain of interaction can be specifically configured to interact with an RTDD and / or with the RT. For example, the interaction domain can comprise intrinsic recognition and DNA binding function without having the same nuclease function to interact specifically with a genomic DNA. In another embodiment, the interaction domain can function as a highly specific interaction partner for an RTDD associated with an RT as further detailed below. Adding this DNA recognition functionality or additional RTDD interaction to the molecular complex by adding an interaction domain, provides another level of specificity to genome engineering in addition to the simple functionality of an isolated SSN.
[00100] Ultimately, particularly the RTDD interacting directly with m RT and additionally the domains of interaction detailed below here, in this patent application, provide a versatile toolkit for (i) bringing a RT in close contact with an SSN from interest and therefore in close proximity to the double chain break induced by at least one SSN to provide a molecular system in the form of an artificial molecular complex having (ii) a higher targeting range and
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72/202 greater precision suitable for a variety of customized genome engineering approaches in eukaryotes and prokaryotes to obtain optimized results for genome engineering, metabolic engineering, plant characteristic development and for therapeutic applications.
[00101] The various aspects and modalities of the present invention are therefore all based on the provision of a suitable double strand-breaking enzyme, or two nickases, as SSN as well as a conveniently designed repair model (RT) nucleic acid sequence , in which the essence of the present invention is the fact that SSN and RT are brought in close proximity to run a genome engineering event in a targeted manner. [00102] In one embodiment, at least one RTDD is a CRISPR gRNA or gDNA and is interacting directly with or is associated with a repair model to build a hybrid RNA-DNA or DNA nucleic acid sequence from RTDD and DNA from the RT.
[00103] An artificial molecular complex according to the present invention therefore represents a complex comprising at least one amino acid component, i.e., an SSN and optionally an interaction domain, an RTDD and a nucleic acid repair (RT) model -basic. In the assembled state, the complex will generally comprise at least one component comprising amino acid (protein), that is, at least one SSN, and one component comprising nucleic acid, that is, RT. The at least one RTDD and optionally at least one interaction domain can also comprise amino acids and / or nucleic acids as building blocks, yet due to the functions of said components within the molecular complex, a greater spectrum of molecules, including blocks, is possible synthetic construction
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73/202 or combinations of different biomolecules and / or synthetic molecules. [00104] The artificial molecular complex according to the present invention, therefore, overcomes the disadvantage of oligonucleotide (RT) -enzyme (SSN) conjugates that cannot self-assemble in vivo, thus severely limiting its usefulness for genome editing in vivo adding at least one additional interaction mediation domain, that is, an RTDD and optionally an AI ensuring a tight association of RT and SSN and a perfect assembly of the molecular complex in vivo, or in general under physiological conditions, in vivo and in vitro when working with at least one intact cell carrying a genomic target DNA (genomic, including coding and non-coding regions, including nuclear, plastid and episomic target DNA and epigenetic target sites) of interest to be modified.
[00105] In one embodiment, the artificial molecular complex can be provided and assembled entirely in vivo, for example, providing the constructs necessary to synthesize and subsequently assemble the complex within a host cell. In another embodiment, the artificial molecular complex can be provided as a molecular complex assembled ex vivo, which is then introduced into a host cell of interest in vivo, or which is brought into contact with a molecule of genomic target interest in vitro. . In yet an additional embodiment, parts of the artificial molecular complex can be produced ex vivo and parts can be produced in vivo, for example, after the introduction of a suitable release vector carrying a plasmid for the transcription and / or expression of a component of the artificial molecular complex, and the final artificial molecular complex exercising its function will then assemble in vivo based on the intrinsic recognition function mediated by RTDD.
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74/202 [00106] An interaction or direct interaction between any components of the artificial molecular complex according to the present invention, therefore, implies any interaction or covalent or non-covalent bond between two components of the artificial molecular complex. A covalent bond, at the nucleic acid level, therefore, can imply a phosphodiester bond or a phosphorothioate bond between nucleotides of a nucleic acid molecule. In addition, a covalent bond can be a disulfide bridge between one amino acid and another amino acid and / or a modified nucleic acid molecule, any naturally occurring or artificial covalent bond according to the present invention can still be envisaged. Non-covalent interactions comprise electrostatic interactions, including ionic, hydrogen bonding or halogen bonding, van-der-Waals forces, including dipolodipole, dipole-induced dipole, London dispersion forces, π effects and hydrophobic effects. Notably, more than one type of interaction can be present within the components of the artificial molecular complex according to the present invention. For example, an SSN, for example, a CRISPR nuclease, can interact through one or more non-covalent interactions with a gRNA like RTDD. The RTDD can be covalently linked to an RT repair model. In another embodiment, an Argonaut fusion protein such as SSN can be covalently fused to a single chain variable antibody fragment as an interaction domain (AI). AI can be, among others, specific for fluorescein and, therefore, can interact non-covalently with RTDD fluorescein. Fluorescein and said nucleic acid from the labeled RT repair model can be provided as a synthetic covalent fusion. In another modality, the association of the different components is mediated by non-covalent interactions, for example, by a
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75/202 leucine zipper recognition of a target DNA sequence and / or an aptamer (based on nucleic acid or amino acid) interacting with either SSN or IA. In one embodiment, the RTDD can be an aptamer, for example, a sequence providing the aptamer function in the repair model. In another embodiment, an extension of a guide nucleic acid allowing hybridization with the repair model can function as at least one RTDD. If defining a guide nucleic acid such as RTDD, a similar modality uses more than one RTDD. In yet a further embodiment, the 3 'or 5' end of the guide nucleic acid used for binding to the repair model can be specifically configured to function as RTDD.
[00107] According to the present invention, the different components of the artificial molecular complex can comprise naturally occurring and / or artificial synthetic building blocks.
[00108] A site-specific nuclease (SSN) according to the various modalities of the present invention, or the nucleic acid sequence encoding it, can therefore be any naturally occurring or manipulated nuclease which is capable of recognizing and cleaving DNA in a site-specific manner. Since many SSNs will have a high number of potential dividing sites within an organism or virus genome, SSNs referred to with a defined divination pattern, or designer SSNs with customized divination patterns, are preferred. Therefore SSNs include site-specific nucleases for genome editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), meganucleases (homing), nucleases derived from CRISPR systems, including Cas or Cpf1 nucleases, or nucleases Argonaut as well
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76/202 rare cut endonucleases, or two site-specific cut endonucleases, including a class IIS restriction endonuclease, including Fokl or a variant thereof, or two site-specific cut endonucleases, or a catalytically active variant or fragment thereof , or any variant or catalytically active fragment of the aforementioned SSNs. Therefore, according to the present invention, more than one SSN, or a nucleic acid sequence encoding the same, may be present, considering that the molecules in short are capable of inducing a targeted double DNA strand break, or two strand breaks. consecutive single strands in a target DNA sequence.
[00109] The target DNA sequence according to the present invention can be any region within a double-stranded, genomic or plasmid-based DNA, where a targeted DNA break is induced and is subsequently repaired with the aid of the model repair (RT) according to the present invention. Although a target DNA sequence originates from an endogenous sequence, the editing or engineering of the referred sequence can be performed in vitro presenting the relevant sequence on A comprising the genomic DNA, preferably on a plasmid. In similar embodiments, the target locus of interest may be comprised of a DNA molecule within a cell. The cell can be a prokaryotic cell or a eukaryotic cell, or a viral genome on a plasmid within a prokaryotic cell or a eukaryotic host cell used for the spread of the virus. The cell can be a mammalian cell. The mammalian cell can be a non-human, bovine, porcine, rodent or mouse cell. The cell can be a non-mammalian eukaryotic cell such as a bird, fish or shrimp. The cell can also be a plant cell. The cell
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The vegetable 77/202 can be a crop plant such as cassava, corn, sorghum, wheat, soy, cotton, sugar beet or rice. The plant cell can also be an algae, tree or vegetable. The modification introduced into the cell by the present invention may be such that the cell and the progeny of the cell are altered for improved production of biological products such as an antibody, starch, alcohol or other desired cell production. The modification introduced into the cell by the present invention can be such that the cell and the progeny of the cell include an alteration that modifies the biological product produced. In another embodiment, the target DNA sequence may be an epigenomic locus of interest.
[00110] A genomic complementarity sequence according to the present invention refers to the portion of the sequence of an RT according to the present invention that can be aligned by means of complementary base pairing. The target DNA sequence and the genomic complementarity sequence may therefore overlap or even be the same, but for certain modalities, the sequences referred to may be different, for example, if at least one SSN will have a cut site upstream or downstream of the RT genomic complementarity sequence portion.
[00111] In any of the described modalities, the chain break can be a double chain break, or it can be two single chain breaks.
[00112] In certain embodiments, the SSN component and optionally the AI component of the artificial molecular complex will be released to a host cell or to a test system comprising a genomic region of interest to be modified through a co-release of protein with the repair model oligonucleotide associated or labeled with
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78/202
RTDD in one embodiment, or in another embodiment as a plasmid-based expression of the fusion protein and subsequent exposure to the repair model marked with RTDD. An additional RTDD can be co-released if more than one RTDD is predicted, for example, one RTDD being a guide nucleic acid molecule and another RTDD being a molecule, for example, biotin or a marker, for example, fluorescein, associated with RT. Plasmid-based or vector-based approaches according to the present invention also include those of a stable SSN expressor line and / or IA and / or a fusion protein thereof.
[00113] In one embodiment, the artificial molecular complex comprises two SSN molecules, one SSN being an active nuclease, and the other SSN being a catalytically inactive deficient nuclease molecule, in which the inactive SSN will function as an interaction partner for one RTDD / RT. The configuration of the mentioned artificial molecular complex can reinforce the specificity for certain target DNA sequences of interest.
[00114] In another embodiment, a fusion protein or a non-covalently associated active Cpf 1 and an inactive dCas9 as the interaction domain can be provided as SSN. The gRNA for Cas9 as RTDD can target the repair model or an extension of it, forming a Cpf1-dCas9-RT complex. The crRNA (Cpf 1) targets the genomic locus defined for the double-stranded cut to initiate homology-directed repair (HDR).
[00115] Likewise, a highly active zinc finger protein, a megaTAL or an inactive meganuclease can be used as the interaction domain.
[00116] In an embodiment according to the various aspects of the present invention, the at least one repair model docking domain (RTDD), or the nucleic acid sequence encoding the
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79/202 same, or at least one artificial molecular complex is selected from at least one among biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with at least one repair model nucleic acid sequence, a streptavidin, or a variant thereof, preferably a monomeric streptavidin, an avidin, or a variant of an affinity marker, preferably a streptavidin marker, an antibody, a single chain variable fragment (scFv), a single domain antibody (nanobody), anticalin, an Agrobacterium VirD2 protein or a domain of the same (see, for example, SEQ ID NO: 33), a Picornavirus VPg, a topoisomerase or a domain thereof, a PhiX174 phage protein, a PhiX A * protein, a protein VirE2 eine or a domain thereof, or digoxigenin. Therefore, RTDD can be a naturally occurring molecule or a synthetic molecule that is not restricted to a nucleic acid or an amino acid molecule. Therefore, preferably, RTDD is a specific interaction mediator of the artificial molecular complex of the present invention, which can be designed in a versatile way to couple at least one SSN of interest and at least one repato model specific to a region of genomic complementarity of interest and optionally carrying an insert of interest to be introduced into a target DNA sequence of interest cleaved by at least one SSN. For modalities using CRISPR or Argonaut based SSNs, RTDD can be a guide nucleic acid sequence. An RTDD according to the present disclosure can, therefore, be a molecule belonging to several classes of artificial or natural molecules. RTDD is therefore
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80/202 defined by its ability to interact directly with at least one repair model nucleic acid (RT) sequence and additionally by interacting directly with at least one SSN. RTDD is, therefore, the molecular ligand within the artificial molecular complex providing close physical proximity to RT and SSN and - due to its double interaction with RT and SSN - ensuring the association of the artificial molecular complex in vitro and in vivo through highly specific molecular interactions. For certain modalities, more than one RTDD may be present carrying more than one RT.
[00117] In another embodiment, the artificial molecular complex comprises an interaction domain, in which the at least one interaction domain, or the nucleic acid sequence encoding the same, is selected from at least one of a DNA binding domain. , a streptavidin, or a variant thereof, preferably a monomeric streptavidin, avidin, or a variant thereof, an affinity marker, a biotinylation signal, a biotin acceptor site, a streptavidin marker, an antibody, an single chain variable fragment (scFv), a single domain antibody (nanobody), anticalin, biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with at least one repair model nucleic acid sequence, a VirD2 d protein and Agrobacterium or a domain thereof, a Picornavirus VPg, a topoisomerase or domain thereof, a PhiX174 phage protein, a PhiX A * protein, a VirE2 protein or a domain of the same, or digoxigenin.
[00118] Noteworthy, an RTDD and an interaction domain can
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81/202 be selected from a comparable and overlapping class of molecules due to the fact that the interaction domain is an optional component, which can additionally optimize the specificity or efficiency of an artificial molecular complex according to the present invention. The presence of an interaction domain may be of importance for modalities using artificial molecular complexes, in which no nucleic acid-guided nuclease such as SSN is used, or in which SSN carries one or more mutations, modifying the activity of the SSN, of intrinsic DNA recognition, ligation or divage. In yet another embodiment, the presence of an interaction domain can be favorable to be used in combination with any type of SSN in order to further increase the range of directionality, the efficiency of connection and / or divage, the rate of divage, or the precision of targeting a target DNA sequence of interest, since the interaction domain as an additional component within the artificial molecular complex can add expanded functionality to the complex and, therefore, the scope of its application can be expanded. Particularly for genome engineering in higher eukaryotes comprising complex genomes, the presence of an additional component, that is, the interaction domain, can therefore be extremely important to obtain an improved accuracy of DNA dividing and targeted repair - mediated by the RT of according to the present invention. In certain embodiments, the AI may represent a highly specific binding partner for a molecule partner not involved in genome engineering itself, where the molecule's partner or cognate-binding partner represents an RTDD being associated with a RT. Therefore, the additional level of addition of an IA domain as well as a cognate partner RTDD can add
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82/202 significantly more connection specificity and availability of
RT to the artificial molecular complex in order to improve the outcome of a targeted genome engineering approach.
[00119] The interaction domain (AI) according to the present invention has several functionalities selected from the group consisting of (i) interaction with at least one repair model docking domain; and / or (ii) interaction with at least one repair model nucleic acid sequence; and / or (iii) sequence-specific interaction with genomic DNA. More than one of these features can be unified within a specific AI.
[00120] It may be preferable to use an AI which represents a protein or polypeptide having high specificity and high affinity binding capabilities intrinsic to a cognate linker, for example, a synthetic linker, including fluorescein to a biomolecule, including biotin or digoxigenin and variants thereof, for an aptamer or an antigen / epitope. The term antigen as used here, in this patent application, and as commonly used in the field of immunology refers to an antibody-generating molecule, that is, a substance, which can elicit an adaptive immune response. An antigen is, therefore, a molecule binding to a specific antigen receptor, either a T cell receptor or a B cell receptor or a variant thereof, for example, a nanobody or a single chain variable antibody fragment, bi-specific antibodies such as tandem discFv, a diabody, a tandem tri-scFv (trivalent) or a triabody (trivalent). An antigen is usually a (poly) peptide, but it can also be a polysaccharide or a lipid, possibly combined with a polysaccharide or protein carrier molecule. Mediated by this intrinsic link / recognition property of the AI, an AI of interest can be chosen which will
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83/202 specifically recognize an RTDD in a highly specific manner and the AI can be connected or fused, covalently or non-covalently, to an SSN. The inclusion of a similar AI, therefore, adds an additional level of specificity to the artificial molecular complex of the present invention and ensures that the RT interacting directly with the RTDD will be specifically associated with the SSN-IA complex as mediated by the IA-RTDD association highly specific. Most preferably, the IA and the cognate RTDD have a high affinity constant or binding affinity and therefore a low dissociation constant (Kd) for each other under physiological conditions, ie a Kd value in the range of low μΜ, or preferably nM, and preferably below. The AI can be a monovalent molecule, a divalent molecule, a trivalent molecule or a multivalent molecule having one or more specificities (fragment derived from trivalent antibody), respectively, or having more than one binding site (tetrameric streptavidin). In these modalities, more than one RTDD and / or RT can be present and be presented to at least one SSN with the artificial molecular complex. Preferred are lAs which have low dissociation constants (Kd), that is, in turn, which have a high affinity for their cognate ligand. In general, subpicomolar dissociation constants are rare as a result of non-covalent bond interactions between two molecules, that is, the typical form of interaction between a protein and a ligand. Nevertheless, there are some important exceptions. Naturally occurring biotin and avidin bind with a roughly 10 ^-15 M dissociation constant, which represents such a high affinity and is not suitable for applications, where a reversible bond is desired. Commercial antibodies or scFvs can have Kd values in the range of 10 ^-14 M to 10 ^-6
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M. For the purposes of the present invention, an IA-RTDD pair will therefore have a low dissociation constant, that is, a high affinity.
[00121] Additionally, in certain modalities the AI can interact directly with the RT. When the RT nucleic acid sequence comprises a stretch, for example, a nucleic acid-based aptamer, this sequence can be recognized by a cognate-binding partner, the IA, which can then interact with the RT in a highly specific. In addition, the IA can be a divalent or trivalent or multivalent molecule having more than one binding specificity. A portion of the AI can be configured to interact with the RTDD, and a portion can be configured to interact with the RT, while the AI is associated with the SSN, so that an even firmer association of the RT and the SSN during genome engineering.
[00122] In another embodiment, the AI can be a binding molecule having the ability to sequence-specific interaction with a genomic DNA. This will add more specificity when targeting an artificial molecular complex to a target DNA sequence. In addition, this makes it possible to use modified SSNs so that an SSN with optimized divination activity can be provided, while AI mediates the function of directing the artificial molecular complex to a target DNA sequence with high precision, whereas SSN and / or AI can interact with an RTDD by interacting with and thus presenting the RT to the site, where a double strand break will be induced. In one embodiment, the AI can therefore be a DNA binding domain or DNA binding motif designed to be part of a fusion protein over either the amino terminus or the carboxy terminus of at least one SSN nuclease or a variant thereof. An amino acid-based binder will allow flexibility and avoid
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85/202 steric impediment to DNA binding or nuclease activity. Potential DNA-binding domains can also be Zinc Fingers (Roy et al. 2012), such as a Zn Cys2 / His2 finger (Kubo et al. 1998), TALENs (Hubbard et al. 2015) or Argonauta or Cas9 proteins inactivated cells capable of highly specific DNA binding. Any of these DNA-binding domains will ideally target a sequence outside the sequence of interest flanked by the homology arm to avoid steric impediment to interaction and thus can add another level of specificity to the artificial molecular complex of the present invention.
[00123] In an additional embodiment, more than one AI domain can be used within the artificial molecular complex, that is, an AI used as a high specificity and affinity ligand for an RTDD, and an AI used as an additional DNA binding domain , both LAs being directly in interaction with, that is, being associated covalently or non-covalently with, at least one SSN of the artificial molecular complex.
[00124] In one embodiment, the at least one SSN and / or the at least one IA comprise a biotinylation signal or biotinylation acceptor site or a strep-tag. The relevant signal / site can be biotinylated in vitro or in vivo by endogenous (BirA) or exogenous biotinylation enzymes / agents, or in an in vitro biotinylation step, and the biotinylated signal / site and / or the strep-tag can be then recognized and linked by a streptavidin or avidin, or preferably a modified variant thereof, most preferably a monomeric variant thereof, where streptavidin or avidin or the variant thereof will be associated with an RT of interest . As it is known that avidin interacts nonspecifically with DNA (Morpurgo et al., 2004), modified variants of avidin or more preferably streptavidin or
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86/202 variants of it may be preferred.
[00125] Particularly for SSNs that are not based on guide RNAs / DNAs, the additional binding capacity and therefore the RT targeting capacity of monomeric streptavidin or scFv with a given binding specificity can dramatically increase the range of SSNs suitable for genome engineering, if used in combination with the RTDDs and / or LAs according to the present invention.
[00126] In one embodiment, biotin can be fused to the DNA of the repair model by commercially available kits or as part of a third party synthesis process such as RTDD. The use of a streptavidin or modified avidin sequence ensures that no inter-protein complex formation occurs and a biotinylated repair model DNA is protein bound. The repair model is then linked to streptavidin or any variant of it as an interaction domain (Niemeyer et al. 1999, Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components. Bioconjug Chem 10 (5): 708-719 ), in which the interaction domain is interacting directly with an SSN, for example, providing SSN and streptavidin as a fusion molecule. In another embodiment, the SSN can comprise a biotinylation peptide or signal and biotinylation will proceed in vivo in a host cell. In this modality, streptavidin or avidin, or a variant thereof, can function as the RTDD itself being linked to an RT. An exemplary sequence encoding a monomeric streptavidin (mSA) suitable as an interaction domain or as an RTDD according to the present invention is shown in SEQ ID NO: 34. mSA fused to an SSN can therefore be understood either as an RTDD or as a domain of interaction according to the present invention. In another modality, the
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SSN can carry a strep-tag, the marker being recognized by a streptavidin variant acting as an interaction domain or as RTDD, respectively. Suitable streptavidin or avidin enzymes, or variants thereof, or vectors encoding the same, are available to the person skilled in the art, for example, at IBA Lifesciences (Gottingen, Germany), addgene (Cambridge, MA, USA), Intregrated DNA Technologies ( Coralville, IA, USA), or GeneArt (ThermoFisher; Waltham, MA, USA). Another exemplary sequence for a monomeric streptavidin construct encoding suitable mSA as IA or RTDD according to the present invention is shown in SEQ ID NO: 42.
[00127] Therefore, in some modality, the interaction or attachment or association between RTDD and SSN and / or the interaction domain results from an interaction of a selected link pair between non-covalent interaction of a selected link pair between, but not limited to: biotin-avidin; biotin-streptavidin; biotin-modified forms of avidin; protein-protein; interactions between nucleic acid proteins; ligand-receptor interactions; linker-substrate interactions; antigen-antibody; single chain antigen-antibody; antibody or single chain-hapten antibody; hormone-binding hormoneprotein; receptor-agonist; receptor-receptor antagonist; IgG-protein A; enzyme-enzyme cofactor; enzyme inhibitor enzyme; Single-stranded DNA-VirE2; StickyC-dsDNA; RISC (RNA-induced silencing complex) -RNA; viral-nucleic acid coating protein; variable fragment of single-chain anti-Fluorescein (anti-FAM scFV) -fluorescein; single-chain variable fragment anti-digoxigenin (DIG) (scFv) immunoglobin (DIG-scFv) -digoxigenin (DIG) and VirD2 binding protein from Agrobacterium or any combination or variation thereof. Noteworthy, antibodies and antibody fragments or
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88/202 derivatives such as scFvs, nanobodies or diabodies having customized specificities and high affinities (in the range of pM or even fM) are commercially available, particularly said antibodies or fragments or variants of the same ligand classic dyes, such as fluorescein, or derivatives thereof .
[00128] In one embodiment, the interaction domain according to the present invention is selected from a leucine zipper, an aptamer sequence, dCas9, dCPF1, a meganuclease, a zinc finger, or a TALE construct. In this embodiment, at least one SSN and RT DNA can be brought into direct interaction through an intermediate DNA binding domain or DNA binding motif designed to be part of a fusion protein on or the amino terminus and / or the SSN carboxy terminus. An amino acid-based ligand will allow flexibility and avoid spherical impediment to DNA binding or nuclease activity. Potential DNA binding domains can also be Zinc fingers (Roy et al. 2012, Prediction of DNA-binding specificity in zinc finger proteins. J Biosci 37 (3): 483-491), such as a Zn Cys2 finger / His2 (Kubo et al. 1998, Cys2 / His2 zinc-finger protein family of petunia: evolution and general mechanism of target-sequence recognition. ”Nucleic Acids Research 26 (2): 608-615), TALENs (Hubbard et al. 2015 , Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. ”Nat Methods 12 (10): 939-942) or inactivated Argonaut or Cas proteins capable of binding to highly specific DNA. Any of these DNA-binding domains as interaction domains can additionally help to target a sequence outside the sequence of interest flanked by the homology arm to avoid spherical impediment to the interaction. The mentioned interaction domains can fulfill the function of increasing the binding to the DNA of the artificial molecular complex of
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89/202 the present invention and / or allow the provision of additional docking sites for RTDD / RT binding to provide a highly specific complex suitable for genome engineering.
[00129] According to certain modalities, the at least one SSN according to the present invention can be fused to a DNA binding domain, that is, a protein or a fragment thereof, or a genetic sequence encoding said protein or a fragment of the protein, which binds DNA molecules or binds other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A fusions, DNA binding domain (DBD), fusions of GAL4 DNA-binding domain, and fusions of herpes simplex virus (HSV) protein BP16.
[00130] In certain modalities, there may be more than one RTDD. The first RTDD can be an mSA or a single chain variable fragment (scFv), while the second RTDD can be biotin or the scFv cognate linker. In one embodiment, using a CRISPR or Argonaut based SSN system, the first RTDD is a guide nucleic acid sequence, and the second RTDD is a biotin or fluorescein or any other high affinity binding partner portion chained to a RT, in which a monomeric streptavidin or a scFv or other cognate protein binding partner represents an AI recognizing the second RTDD and bind to it with high affinity. This design of the artificial molecular complex according to the present invention allows maximum flexibility to bring a RT in close contact with an effector SSN while simultaneously providing high availability of RT and no loss of RT, since RTDDs provide strong and reliable interactions with the RT and SSN for precision genome engineering events.
[00131] In one modality, connection of repair model to SSN
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90/202 can be performed by a single chain variable antibody fragment (scFv) against the Fluorescein dye. The scFv specifically binding fluorescein and fluorescein derivatives is fused to SSN in a hybrid protein manner (Schenk et al. 2007, Generation and application of a fluorescein-specific single-chain antibody. Biochimie 89 (11): 1304-1311). In another embodiment, the SSN can comprise a fluorescein molecule interacting with it and the specific cognate fluorescein scFv can be provided as a fusion with an RT and can bind to the fluorescein associated with the SSN. In this way the scFv can function as RTDD or as an interaction domain according to the present invention.
[00132] In other embodiments of the present invention, any scFv with a different binding specificity can be used.
[00133] Suitable scFv-ligand pairs are selected from the group consisting of a scFv and fluorescein (FAM) or any FAM derivative or variant, a scFv recognizing digoxigenin (DIG), a customized scFv recognizing an epitope / antigen on an SSN of interest, and the like. An example of a sequence encoding an scFv encoding sequence capable of binding to fluorescein is shown in SEQ ID NO: 43.
[00134] In another embodiment, an aptamer sequence is designed to interact specifically with at least one SSN. In this embodiment, the aptamer sequence can be covalently or non-covalently linked to a repair model sequence of interest to allow a direct association between the SSN and the aptamer as RTDD without creating a fusion protein and / or using an additional interaction domain. In modalities, in which no separate interaction domain is used, the RTDD interacting with the RT comprises a nucleotide motif capable of interacting
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91/202 specifically, that is, attaching or linking to a domain of at least one SSN protein, or to a specific domain of the same configured to interact with RTDD: In some modalities, the interaction is selected between, but not limited to: Protein Zinc Finger-Motive Zinc Finger; restriction enzyme recognition domain restriction enzyme recognition sequence; DNA binding domain of transcription factor-DNA motif; repressor-operator; leucine zipper promoter; Helix domain Helix loop-E box; RNA binding motifs comprising Arginine-Rich Motif domains, αβ protein domains, RNA Recognition Motif (RRM) domains, K-Homology Domains, Double-stranded RNA-binding motifs, Zinc-binding fingers RNA, and RNA sequence-targeting enzymes-cognate specific RNA; HlV-rev protein-HB of the HIV rev response element (RRE); Tat major binding domain of bovine immunodeficiency virus (BIV) -loop 1 of the BIV trans-action response element (TAR) sequence; White phage, phi21 proteins, and P22 N, boxB loop hairpins at the N-use (nut) sites in their respective RNAs.
[00135] As far as the present invention refers to the use of an Argonaut as a site-specific nuclease, in addition to the advantages of a guide DNA molecule, the release of the NgAgo endonuclease is facilitated by its small size. Wild protein (WT) (GenBank Accession number AFZ73749) has 887 amino acids, or roughly 2/3 the size of the Streptococcus pyogenes Cas9. This simplifies the cloning and assembly of the vector, can increase the levels of nuclease expression in cells, and reduces the challenge in expressing the protein from highly size-sensitive platforms, such as viruses, including either DNA or RNA viruses. Like other nucleic acid-guided endonucleases, NgAgo SSNs
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92/202 generally require a minimum of two components for targeted mutagenesis in plant cells: a single 5'-phosphorylated guide DNA and the endonuclease protein NgAgo. For targeted editions, insertions, or sequence substitutions, a DNA model encoding the desired sequence modifications can also be provided for the plant cell to introduce guide DNA either through the NHEJ or HR repair pathways. The success of editing events is most commonly detected by phenotypic modifications (such as by knockout or introduction of a gene that results in a visible phenotype), by PCR-based methods (such as by enrichment PCR, digestion by PCR, or endonuclease tests (T7EI or Surveyor), or by sequencing the next targeted generation (NGS, Next Generation Sequencing; also known as deep sequencing). In a specific embodiment, the modified Argonaut endonuclease is active at a temperature from about 200 to about 350. In a specific embodiment, the modified Argonaut endonuclease is active at a temperature from about 230 to about 320 Argonaut proteins which can function as endonucleases can comprise three key functional domains: a PIWI endonuclease domain, a PAZ domain, and a MID domain. The PIWI domain can look like a nuclease. The nuclease can be an RNase H or a DNA-guided ribonuclease. The PIWI domain can share a divalent cation binding motif for catalysis presented by other nucleases that can cleave RNA and DNA. The divalent cation binding motif may contain four negatively charged evolutionary conserved amino acids. The four negatively charged evolutionary conserved amino acids can be aspartate-glutamate-aspartate-aspartate (DEDD). The four evolutionary conserved amino acids
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Negatively charged 93/202 can form a catalytic tetrad that binds two Mg2 + ions and cleaves a target nucleic acid in products carrying a group of 3 'hydroxyl and 5' phosphate. The PIWI domain can additionally comprise one or more amino acids selected from a basic residue. The PIWI domain can additionally comprise one or more amino acids selected from histidine, arginine, lysine and a combination thereof. Histidine, arginine and / or lysine can play an important role in catalysis and / or divage. Divination of the target nucleic acid by Argonaut can occur in a single phosphodiester bond. In some cases, one or more magnesium and / or manganese cations can facilitate the dividing of the target nucleic acid, in which a first cation can attack nucleophilically and activate a water molecule and a second cation can stabilize the transition state and the group of departure. For certain Argonauta nucleases, the length of the gDNA will provide the affinity between the Argonauta and the guide gDNA.
[00136] Suitable argonaut proteins according to the present invention are shown with SEQ ID NOs: 19 and 20, or can comprise a sequence having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology with the same as long as the homologous sequence still fulfills the function of argonaut protein from which it is derived, that is, from which it originates. Additional suitable argonaut sequences are disclosed in United States Provisional Patent Application No. U.S. 62 / 345,448 which are hereby incorporated into this patent application by reference. Additional suitable argonaut sequences can be derived from a sequence according to SEQ ID NOs: 21 to 29 or a sequence
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94/202 having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence homology with them.
[00137] An Argonaut can comprise a nucleic acid binding domain. The nucleic acid binding domain can comprise a region that contacts a nucleic acid. A nucleic acid binding domain can comprise a nucleic acid. A nucleic acid binding domain can comprise a proteinaceous material. A nucleic acid binding domain can comprise nucleic acid and a proteinaceous material. A nucleic acid binding domain can comprise DNA. A nucleic acid binding domain can comprise single-stranded DNA. Examples of nucleic acid binding domains may include, but are not limited to, a helix-loop-helix domain, a zinc finger domain, a leucine zipper domain (bZIP), a winged helix domain, a winged helix-loop-helix domain, a helix-loop-helix domain, an HMG-box domain, a Wor3 domain, an immunoglobulin domain, a B3 domain, or a TALE domain. A nucleic acid binding domain can be an Argonaut protein domain. An Argonaut protein can be a eukaryotic Argonaut or a prokaryotic Argonaut. An Argonaut protein can bind to RNA or DNA, or both RNA and DNA. An Argonaut protein can cleave RNA, or DNA, or both RNA and DNA. In some cases, an Argonaut protein binds to DNA and cleaves the DNA. In some cases, the Argonaut protein binds to double-stranded DNA and cleaves double-stranded DNA. In some cases, two or more nucleic acid binding domains can be linked together. Linking a plurality of nucleic acid binding domains together can provide increased
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95/202 polynucleotide targeting specificity. Two or more nucleic acid binding domains can be linked via one or more ligands. The binder can be a flexible binder. Binders can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 30, 35, 40 or more amino acids in length. The binding domain may comprise glycine and / or serine, and in some embodiments it may consist of or may consist essentially of glycine and / or serine. Ligands can be a nucleic acid ligand which can comprise nucleotides. A nucleic acid ligand can link two DNA binding domains together. A nucleic acid ligand can be a maximum of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. A nucleic acid ligand can be at least 5, 10, 15, 30, 35, 40, 45, or 50 or more nucleotides in length. Nucleic acid binding domains can bind to nucleic acid sequences. Nucleic acid binding domains can bind to nucleic acids through hybridization. Nucleic acid binding domains can be manipulated (for example, manipulated to hybridize to a sequence in a genome). A nucleic acid binding domain can be manipulated by molecular cloning techniques (for example, targeted evolution, site-specific mutation, and rational mutagenesis).
[00138] In certain embodiments, the SSN according to the present invention will be a CRISPR nuclease, including Cas or Cpf 1, or an Argonauta nuclease, or a catalytically active variant or fragment thereof. Suitable CRISPR nuclease sequences are selected from the group consisting of SEQ ID NOs: 19 to 29, or 35 to 41 or a sequence having at least 66%, 67%, 68%, 69%, 70%, 71%, 72% , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%,
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91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology with the same. Additional suitable Cas or Cpf 1 effectors can be derived from an organism of a genus comprising Streptococcus, Campylobacter, Candidatus Micrarchaeum acidiphilum ARMAN-1, Parcubacteria (GenBank: APG80656.1), Sulfolobus spp., Including Sulfolobus islandicus HVE10 / 4 ( GenBank: ADX81770.1) or REY15A (GenBank: ADX84852.1), Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynobacterium, Chrysobacteria, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, eg. , S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii.
[00139] In one embodiment, at least one site-specific nuclease or the catalytically active fragment thereof as part of the artificial molecular complex of the present invention, or the sequence encoding it, is independently selected from the group consisting of a Cas polypeptide of Streptococcus spp., including Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria spp., including Neisseria meningitides, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Flactobacillus, Bactero, Bacteria,
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Gluconacetobacter, Roseburia, Parvibaculum, Nitratifractor, Mycoplasma and Campylobacter, Candidatus Micrarchaeum acidiphilum ARMAN-1, Parcubacteria (GenBank: APG80656.1), Sulfolobus spp. ADX84852.1), a Cpf1 polypeptide from an archaea or a bacterium, including a Cpf1 polypeptide from Acidaminococcus spp., Including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., Including Lachnospiraceae bacterium ND2006, Lachnospiraceae bacterium MC2017, Lachnospiraceae bacterium MA2020, Butyrivibrio proteoclasticus, Candidatus spp. SCADC, Smithella sp. SC_K08D17, Francisella spp., Including Francisella novicida U112, Eubacterium eligens, Prevotella spp., Or Porphyromonas spp., Or an Argonauta nuclease of Natronobacterium gregoryi (GenBank: AFZ73749.1), Microcystis aeruginosa (Reference sequence NCBI: WP_0.19 NCBI Reference String:
WP_002747795.1 or NCBI Reference String:
WP_012265209.1), Halogeometricum pallidum (GenBank: ELZ29017.1), Natrialaba asiatica (Reference Sequence NCBI: WP_006111085.1), Natronorubrum tibetense (Reference Sequence NCBI: WP_006090832.1), Natrinema pellirubrum18 .1), or Synechococcus spp. (NCBI Reference Sequence: WP_011378069.1) or variants and / or functional fragments and / or combinations thereof, including nickases, or nucleases lacking endonucleolytic activity.
[00140] In additional embodiments of the invention using at least one Cpf1 effector as SSN, an adjacent motif of
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98/202 protospacer (PAM) or motif similar to PAM directs the binding of the effector protein complex to the target locus of interest. In one embodiment using at least one Cpf 1 effector as SSN, PAM is 5 'TTN, where N is A / C / G or T. In another preferred embodiment of the invention, PAM is 5' TTTV, where V is A / C or G. In certain embodiments, the PAM is 5 'TTN, where N is A / C / G or T and the PAM is located upstream of the 5' end of the protospace. In certain embodiments of the invention, the PAM is 5 'CTA, and the PAM is located upstream of the 5' end of the protospace or target locus. In certain modalities, an expanded targeting range is provided for RNA-guided genome editing nucleases where the T-rich PAMs of the Cpf1 family enable the targeting and editing of AT-rich genomes.
[00141] In certain embodiments, the CRISPR enzyme is produced by genetic engineering and can comprise one or more mutations that reduce or eliminate a nuclease activity. Likewise, the present invention contemplates a method of using two or more nickases, in particular a dual or double nickase approach to generate a targeted double DNA strand break. [00142] In embodiments using Cpf 1 effector protein complexes within the artificial molecular complex according to the present invention, a Cpf1 effector may be used having one or more naturally occurring or manipulated or modified or optimized nucleic acid components, or the encoded protein. In a preferred embodiment the nucleic acid component of the complex may comprise a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem loops or optimized secondary structures. In a preferred embodiment, the direct repeat has a minimum length of 16 nucleotides and a single stem loop.
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In additional embodiments the direct repetition has a length greater than 16 nucleotides, preferably more than 17 nucleotides, and has more than one stem loop or optimized secondary structures. In a preferred embodiment, the direct repeat can be modified to comprise one or more protein-binding RNA aptamers. In a preferred embodiment, one or more aptamers can be included as part of the optimized secondary structure. The aforementioned aptamers may be capable of binding to a bacteriophage coating protein. The bacteriophage coating protein can be selected from the group comprising Οβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, Fl, ID2 , NL95, TW19, AP205, (pCb5, (pCb8r, <pCb12r, (pCb23r, 7s and PRR1. In a preferred embodiment the bacteriophage coating protein is MS2.
[00143] In certain embodiments, the invention also provides for one or more mutations or the two or more mutations to be in a catalytically active fragment of at least one SSN effector protein comprising a RuvC domain. In some embodiments, the RuvC domain may comprise a RuvCI, RuvCII or RuvCIII domain, or a catalytically active domain which is homologous to a RuvCI, RuvCII or RuvCIII domain, etc. or any relevant domain as described in any of the methods described here , in this patent application. The SSN effector protein can comprise one or more heterologous functional domains. The one or more heterologous functional domains of the artificial molecular complex may comprise one or more domains of nuclear localization signal (NLS). The one or more heterologous functional domains may comprise at least two or more NLS domains. The one or more NLS domains can be positioned at or near or in promixity at the end of the
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100/202 effector protein (for example, Cpf1) and in the case of two or more NLSs, each of the two can be positioned at or near or at a termination of the effector protein (for example, Cpf1). The one or more heterologous functional domains may comprise one or more domains of transcriptional activation. In a preferred embodiment, the transcriptional activation domain can comprise VP64. The one or more heterologous functional domains may comprise one or more domains of transcriptional repression. In a preferred embodiment, the transcriptional repression domain comprises a KRAB domain or a SID domain (for example, SID4X). The one or more heterologous functional domains may comprise one or more nuclease domains as SSNs. In one embodiment, the SSN can comprise Fok1 or a catalytically active fragment or variant thereof.
[00144] In a preferred embodiment, at least one site-specific nuclease or the catalytically active variant or fragment thereof of the artificial molecular complex according to the present invention, or the sequence encoding it, is selected from a CRISPR nuclease, preferably between a Cas nuclease or a Cpf1, or a Fokl nuclease, or a catalytically active fragment thereof, and the at least one interaction domain, or the sequence encoding the same, is selected from a single chain variable fragment or a monomeric streptavidin.
[00145] In one embodiment, the artificial molecular complex according to the present invention comprises at least one CRISPR or SSN derived from Argonaut, or a variant or catalytically active fragment thereof, and at least one guide nucleic acid sequence representing the at least one repair model docking domain, where each of the at least
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101/202 minus a guide nucleic acid sequence comprises (i) a first portion of the sequence that is complementary to a target recognition DNA sequence, and (ii) a second portion of the sequence, wherein the second portion of the sequence is configured to interact with at least one site-specific nuclease, and (iii) wherein at least one guide nucleic acid sequence is physically associated with at least one repair model nucleic acid sequence and therefore forms a sequence hybrid nucleic acid comprising or consisting of at least one RNA or DNA and at least one additional DNA nucleic acid sequence, and (iv) optionally comprising a linker region between at least one guide nucleic acid sequence and at least a repair model nucleic acid sequence, preferably where the repair model nucleic acid sequence is associated with the gu nucleic acid sequence ia at the 3 'end of the guide nucleic acid sequence, and / or where the repair model nucleic acid sequence is associated with the 5' end of the guide nucleic acid sequence, and / or where the nucleic acid sequence repair model is located within the guide nucleic acid sequence.
[00146] The at least one repair template nucleic acid sequence and / or at least one guide nucleic acid sequence according to the various aspects and embodiments of the present invention comprises a nucleotide sequence selected from a nucleotide sequence that occurs naturally or not naturally, including a synthetic nucleotide sequence, optionally comprising backbone and / or base modifications, wherein the guide nucleic acid sequence comprises a single or partially single-stranded RNA or DNA nucleotide sequence , and in which at least one sequence
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Repair model nucleic acid 102/202 comprises a single-stranded or a double-stranded DNA nucleotide sequence.
[00147] In certain embodiments, the at least one repair model (RT) nucleic acid sequence of the artificial molecular complex according to the present invention comprises at least one end portion, preferably the 3 'end, wherein this end portion does not interact with any other component of the artificial molecular complex and is, therefore, configured to hybridize to at least one genomic complementarity sequence in order to mediate repair of the target DNA sequence, and / or where at least a repair model nucleic acid sequence is provided as a plasmid. In order to be able to access at least one genomic complementarity sequence, the RT will therefore be provided in a configuration allowing optimal base pairing with at least one genomic complementarity sequence. This configuration will vary depending on the nature of the RT and depending on how the RT is provided. In certain embodiments, at least one RT is used which can be attached covalently or non-covalently to an RTDD, for example, a gRNA or a gDNA.
[00148] In certain modalities using a molecule comprising at least a stretch of RNA as RTDD, or using RNA encoding a protein of interest, the RNA can be presented together with a molecule or protective or protective chain, whose protective molecule will ring through less partially to RNA representing the actual effector molecule of the artificial molecular complex to protect the RNA effector molecule from degradation within the cell.
[00149] Suitable configurations for a molecular complex
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Artificial 103/202 according to the present invention are shown in Figures 1 to 4. Artificial molecular complexes using a hybrid nucleic acid sequence such as RTDD and RT according to the present invention are shown in Figure 1 A to D and Figure 2 A to C, but are not restricted to them. Depending on the SSN, the repair model (RT) may be in a form of ssDNA or dsDNA and, if a CRISPR or Argonaut protein is used as SSN, it can be attached to at least one guide nucleic acid (sgRNA or gRNA or gDNA) at the 3 'end, at the 5' end in a covalent or non-covalent mode or may be located within the gRNA, for example, forming a secondary hairpin structure of a defined size and shape. This design allows both the gRNA and the RT portion to both perform their functions without disturbing the interaction of at least one gRNA of interest with a CRISPR or Argonaut nuclease of interest and simultaneously positioning the RT in close proximity to the site of a DNA breakage induced by at least one pair of CRISPR / Argonaut gRNA / gDNA.
[00150] In certain embodiments using CRISPR nucleases, the artificial molecular complex will comprise a hybrid nucleic acid sequence comprising or consisting of at least one RNA and at least one DNA nucleic acid sequence or simply an RNA / nucleic acid sequence Hybrid DNA according to the present invention, therefore, represents a molecule comprising RNA and chimeric DNA, which comprises two functionalities. First, it comprises a portion of guide nucleic acid (gRNA), comprising a ribonucleic acid. This gRNA comprises two portions of a nucleotide sequence, a nucleotide sequence being necessary for interaction with a CRISPR polypeptide of interest as well as another sequence of
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104/202 nucleotides comprising a targeting domain, where the targeting domain is able to hybridize through base pairing to a complementary DNA target sequence of interest adjacent to a PAM sequence on the opposite strand, this complementary DNA target sequence therefore representing the first target DNA sequence according to the present invention. Second, the hybrid RNA / DNA nucleic acid sequence comprises a repair model nucleic acid sequence which may comprise a desired edition to be introduced into a target DNA sequence of interest. Furthermore, the repair model nucleic acid sequence may comprise additional homologous sequence immediately upstream and downstream of the target DNA sequence, i.e., branches of homology to the left and right. The length and connection position of each branch of homology is dependent on the size of the modification being introduced, and can be adjusted for optimal efficiency. For example, it is likely that a repair model with specific complementarity for the cleaved DNA chain first released by Cas9 (as described in Richardson, et al., Nature Biotechnology. 2016, doi: 10.1038 / nbt.3481) can produce the repair more efficient. The repair model can be a nucleotide sequence of single-stranded DNA or double-stranded DNA depending on the specific application.
[00151] The repair model may contain polymorphisms in relation to genomic DNA to break the bond by the nuclease, otherwise the repair model becomes an adequate target for dividing the CRISPR polypeptide. For example, PAM can be mutated in such a way that it is no longer present, but the coding region of the gene is not affected, which corresponds to a silent mutation that does not modify the encoded amino acid sequence. In another
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105/202 modality, where a nuclease-deficient CRISPR polypeptide is used within the artificial molecular complex as SSN, the presence of a PAM sequence within the repair model sequence is possible. In one embodiment, the RTDD / RT sequence comprises at least one guide nucleic acid sequence and at least one repair model nucleic acid sequence, but the RTDD / RT hybrid can also comprise additional portions attached to it suitable for editing. genome as further detailed below. In another embodiment the hybrid RTDD / RT sequence consists of at least one guide nucleic acid sequence and at least one repair model nucleic acid sequence.
[00152] It has been seen that there can be an optimal RT size depending on the SSN used that provides a balance of nuclease efficiency with the size of the homology arm for the efficiency of HR-mediated DSB repair.
[00153] In one embodiment, the guide nucleic acid sequence or gRNA is provided as an RNA nucleic acid sequence unifying a tracrRNA element and a crRNA element. In another embodiment, for example, when working with a Type V CRISPR system using a Cpf 1 polypeptide or a catalytically active variant or fragment thereof, the gRNA comprises a crRNA element. In yet a further embodiment, the gRNA can be provided as more than one RNA nucleic acid sequence simulating the natural situation in many CRISPR systems in which crRNA and tracrRNA, if both are needed, are provided on two separate RNA molecules. In certain modalities, this arrangement therefore allows the possibility of having the two elements (tracrRNA and crRNA) in separate RNA chains as in nature. In one mode, it is
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106/202 a separate RNA nucleic acid molecule is provided providing a crRNA and a separate RNA nucleic acid molecule is provided, i.e., more than one RTDD is provided. Either the crRNA portion or the tracrRNA portion can be associated with a repair template (RT) nucleic acid sequence. For example, it may be preferable to provide a tracrRNA: RT hybrid or a crRNA: RT when ex vivo chemical synthesis of tracrRNA: RT or crRNA: RT is chosen due to the shorter length of the respective molecule compared to a gRNA: RT hybrid, where gRNA consists of a single RNA molecule unifying the function of crRNA and tracrRNA.
[00154] The RTDD / RT sequence according to the present invention is therefore suitable for precision genome editing on any cell type of interest, including prokaryotic cells and eukaryotic cells, including fungal, animal and plant cells and for any genome of interest in an in vitro configuration and represents a physically connected tool suitable to allow simultaneous space-time availability of a repair model and SSN during genome editing.
[00155] According to all aspects and modalities of the present invention, the at least one RTDD and at least one repair model nucleic acid sequence are associated with each other. The term associated with or in association in accordance with the present disclosure should be considered in general and therefore in accordance with the present invention implies that an RTDD, for example, a gRNA or a biotin molecule, FAM or a digoxigenin, it is provided in physical association with a DNA repair model, the association being either covalent or non-covalent in nature, inherently increasing the availability of the repair model for homologous recombination. Rather than
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107/202 indiscriminate amplification of the repair model, or excess provision of the repair model, but physically not linked to RTDD, the nucleotide sequence of the repair model is therefore presented in the DSB together with the SSN of the artificial molecular complex for a target DNA sequence of interest, which in turn significantly increases the predictability and specificity of a genome editing approach.
[00156] In an additional embodiment according to the present invention, at least one repair model nucleic acid sequence is attached to at least one RTDD sequence by means of both covalent and / or non-covalent bonds or attachments. According to this modality, the hybrid complex of RTDD and RT can be provided as a molecule synthesized in vitro which can then be associated with at least one SSN of interest, either in vitro, or in vivo in the target cell of interest, or within an in vitro assay of interest. Preferably, the cell is a eukaryotic cell, including a fungal cell, an animal cell or a plant cell. The cell can also be a prokaryotic cell. In addition, the cell can be a prokaryotic or eukaryotic host cell carrying, either on a plasmid or integrated within the genome, a heterologous target sequence from another organism or virus. In this embodiment, the cell functions as a host to perform genome engineering on a heterologous sequence provided within the said host cell.
[00157] In an embodiment according to the various aspects of the present invention at least one repair model (RT) nucleic acid sequence is covalently attached to at least one RTDD. A covalent attachment or covalent bond is a chemical bond that involves the sharing of electron pairs between atoms of the molecules or sequences attached so
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108/202 covalent to each other.
[00158] In another embodiment according to the various aspects of the present invention at least one repair model nucleic acid sequence is non-covalently attached to at least one RTDD sequence. A non-covalent interaction differs from a covalent bond in that it does not involve electron sharing, but on the contrary it involves more dispersed variations of electromagnetic interactions between molecules / sequences or within a molecule / sequence. Non-covalent interactions or attachments therefore comprise electrostatic interactions, van der Waals forces, TT effects and hydrophobic effects. Of particular importance in the context of nucleic acid molecules are hydrogen bonds as an electrostatic interaction. A hydrogen bond (H hydrogen) is a specific type of dipole-dipole interaction that involves the interaction between a partially positive hydrogen atom and an oxygen, nitrogen, sulfur, or highly electronegative, partially negative fluorine not bound in any way covalent to the referred hydrogen atom.
[00159] The term hybridization as used here, in this patent application, refers to the pairing of complementary nucleic acids, that is, DNA and / or RNA, using any process by which a nucleic acid chain is joined with a complementary chain through base pairing to form a hybridized complex. Hybridization and the strength of hybridization (that is, the strength of the association between nucleic acids) is impacted by factors such as the degree and length of complementarity between nucleic acids, the severity of the conditions involved, the Tm of the hybrid formed, and the proportion of G: C within nucleic acids. The term hybridized complex refers to a complex formed between two nucleic acid sequences due to the formation of
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109/202 hydrogen bonds between complementary G and C bases and between complementary A and T / U bases. A hybridized complex or a corresponding hybrid construct can be formed between two DNA nucleic acid molecules, between two RNA nucleic acid molecules, or between a DNA nucleic acid molecule and an RNA nucleic acid molecule. For all constellations, the nucleic acid molecules can be naturally occurring nucleic acid molecules generated in vitro or in vivo and / or artificial or synthetic nucleic acid molecules. Hybridization as detailed above, for example, WatsonCrick base pairs, which can form between DNA, RNA and DNA / RNA sequences, are dictated by a specific hydrogen bonding pattern, which therefore represents a form of non-covalent attachment according to the present invention.
[00160] With respect to non-covalent associations according to the present invention, the at least one RTDD of the artificial molecular complex of the present invention and at least one repair model sequence of the present invention can be associated with each other by pairing of RNA-DNA bases.
[00161] Another form of non-covalent interaction is the association of at least one repair model sequence with at least one component, either RTDD or an RTDD understood by SSN, by electrical charges.
[00162] With respect to covalent attachment or attachment, the at least one RTDD and at least one repair model sequence are connected as contiguous molecules, whether produced in vivo or in vitro. Covalent and non-covalent attachment can also be combined, for example, by providing a covalently attached RTDD / repair model sequence, which may additionally comprise a nucleic acid sequence of
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110/202 additional repair model attached non-covalently to the RTDD / repair model sequence covalently attached. This approach is especially suitable if the RTDD / covalently attached repair model sequence is at least partially produced in vivo and an additional repair model, whether produced in vivo or in vitro, has to be added to the RTDD complex / pre-existing repair model.
[00163] As is also evident from Nishimasu et al., Supra, a gRNA is configured to interact with a CRISPR polypeptide or a catalytically active variant or fragment thereof according to the disclosure of the present invention, if the gRNA comprises at least a portion usually comprising a heteroduplex configuration, which is recognized by a CRISPR polypeptide either in a sequence-dependent manner, that is, through interaction with the bases of an RNA, comprising A, U, G and C, or in a sequence independent manner, that is, through phosphate interaction of the structure of a gRNA nucleotide sequence with a CRISPR polypeptide.
[00164] According to certain modalities of the first aspect as well as of the later aspects of the present invention, the target DNA sequence is located within the genome of a cell, preferably a prokaryotic cell or eukaryotic cell, more preferably a cell fungal, an animal cell or a plant cell, where the genome comprises the nuclear genome as well as other parts of the genome, including the plastid genome.
[00165] A target DNA sequence defines the genomic region, where targeted genome editing should be done. Due to the fact that the RTDD and the repair model nucleic acid sequence have intrinsically different functionalities, there may be
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111/202 more than one target region of DNA, which may be different for the different components of the artificial molecular complex of the present invention. A target DNA sequence can therefore define the region of a target DNA region of interest which is complementary to the sequence, the portion of RTDD or RTDD being a gRNA, while another target DNA sequence defines the region of a target DNA region of interest to which an SSN and / or an interaction domain will connect. At least a portion of the repair model nucleic acid sequence is complementary to and is defined as a genomic complementarity sequence, said sequence also representing an additional DNA target sequence. The target DNA regions can be the same, or preferably different, yet possibly overlapping regions, within the target DNA sequence of interest.
[00166] The spatial relationship between the target site of an RTDD and / or SSN and / or an interaction domain and the homology site for the repair model nucleic acid (RT) sequence can be variable. The two sites can be identical, can be completely or partially overlapping, or can be separated by any number of nucleotides within the genome of interest. RT can have homology for both strands of genomic DNA, optionally presented as a double-stranded construct, for example, a plasmid, or any strand individually, regardless of which strand is targeted. An efficient repair model can be configured to have specific complementarity to the first cleaved DNA strand released by an SSN, for example, a Cas9 (as described in Richardson, et al., Nature Biotechnology. 2016, doi: 10.1038 / nbt. 3481).
[00167] The interaction between RTDD and RT and, therefore, the close proximity of SSN and RT according to the molecular complex
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The artificial 112/202 of the present invention is expected to overcome the generally low homology-directed repair (HDR) / homologous recombination (HR) efficiency since it guarantees the physical availability of the nucleotide sequence of the repair model present in a stoichiometric mode in with respect to at least one SSN in situ at the site where a genomic chain break targeted by at least one SSN polypeptide is introduced into a target DNA sequence.
[00168] The term repair model (RT) nucleic acid sequence as part of the artificial molecular complex according to the present invention therefore implies a nucleotide sequence, which can be a single-stranded or double-stranded DNA sequence , which is capable of providing a model for modifying and / or repairing a DNA break.
[00169] In an embodiment according to the present invention, the artificial molecular complex is a pre-assembled complex in vitro, in which the SSN, RTDD and RT and optionally the component or portion of the interaction domain are provided either attached covalently to each other or non-covalently associated. In one embodiment, the RTDD / RT sequence is pre-assembled and the SSN and optionally the interaction domain are released separately within a target cell, either as transcribable DNA or as a translatable RNA construct or directly as an amino acid sequence and the sequence RTDD / RT and the SSN and optionally the interaction domain form a complex within the target cell. In another embodiment, the RTDD / RT sequence as well as the SSN and optionally the interaction domain are assembled in vitro and the nucleoprotein complex optionally comprising additional molecules, for example, biotin or FAM, or digoxigenin, is then introduced into a target cell of interest or
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113/202 within an in vitro system comprising at least one target DNA nucleotide sequence of interest to be modified. [00170] The introduction of a functional pre-assembled artificial molecular complex within a target cell results in a targeted double chain break and simultaneous site-specific repair and modification due to the fact that the activity of at least one site-specific nuclease (SSN) is immediately accompanied by subsequent homologous recombination at the target DNA sequence site according to the present invention with the DNA repair model nucleic acid sequence linked to RTDD, RTDD also interacting directly with an SSN. Therefore, the disadvantages of low availability of an RT or non-specific NHEJ events (See Background of the Invention above) hindering a highly specific and controllable genome editing event can be simultaneously reduced, since the artificial molecular complex can reach a site target in a coordinated manner in an appropriate stoichiometric composition of repair and nuclease model. An additional benefit is that the potential for integrating the off-target repair model is reduced due to its physical association with the protein as well as with the complex's RTDD, where SSN and / or RTDD cannot be integrated within the perse genome.
[00171] The term homology-directed repair in accordance with the present disclosure comprises any type of changes that may be introduced by the repair model sequence in accordance with this requirement, which may independently comprise sequence inserts, editions of at least one sequence position, deletions or rearrangements, the preferred strategy for genome editing approaches in larger eukaryotes currently being insertions, deletions
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114/202 or editions, since these strategies allow the targeted knock-in or knock-out of a sequence of interest within a target DNA sequence, or a site-specific modification of at least one sequence.
[00172] An example for targeted repair by targeted homology as mediated by an artificial molecular complex using a CRISPR nuclease as SSN formed ex vivo or in vivo in cooperation with the hybrid nucleic acid sequence according to the present invention can be found in Figure 3 A to E illustrating the chronological sequence of subsequent DNA recognition, ligation, divination and repair for an example of an SSN / guide nucleic acid complex (RTDD) / repair model (RT) and for a given endogenous DNA target sequence.
[00173] In an embodiment according to the various aspects of the present invention, the repair model nucleic acid sequence and / or at least one RTDD sequence comprises a nucleotide sequence selected from a naturally occurring nucleotide sequence or not naturally, including a synthetic nucleotide sequence, optionally comprising backbone and / or base modifications, wherein the guide nucleic acid sequence comprises a single or partially single-stranded RNA nucleotide sequence, and where the The repair model nucleic acid sequence comprises a single-stranded or a double-stranded DNA nucleotide sequence.
[00174] A challenge for any CRISPR genome editing approach is the fact that the gRNA and the functional CRISPR polypeptide as SSN must be transported to the nucleus or any other compartment comprising genomic DNA, that is, the target sequence of DNA, in a functional (not degraded) mode.
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As RNA is less stable than a double-stranded polypeptide or DNA and has a longer turnover, especially as it can be easily degraded by nucleases, in some embodiments according to the first aspect of the present invention, gRNA as RTDD and / or a DNA repair model nucleic acid sequences comprise at least one naturally occurring nucleotide. Preferred modifications of the structure according to the present invention increasing the stability of the gRNA and / or the DNA repair model nucleic acid sequence are selected from the group consisting of a phosphorothioate modification, a methyl phosphonate modification, a modification of blocked nucleic acid, a 2O- (2-methoxyethyl) modification, a di phosphorothioate modification, and a peptide nucleic acid modification. Noteworthy, all of the aforementioned structural modifications still allow the formation of complementary base pairing between two nucleic acid chains, they are even more resistant to dividing by endogenous nucleases. Depending on the nuclease used in accordance with the present invention, it may be necessary not to modify the nucleotide positions of a gRNA, which are involved in sequence-independent interaction with the CRISPR polypeptide. The referred information can be derived from the structural information available as available for CRISPR / gRNA nuclease complexes.
[00175] In certain embodiments according to the first aspect of the present invention, the RTDD and / or the DNA repair model RT nucleic acid sequence and / or the interaction domain comprises / comprises a modification of nucleotide and / or base, preferably at selected positions in the nucleotide sequence, not all. These modifications are selected from the group consisting of addition
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116/202 acridine, amine, biotin, cascade blue, cholesterol, Cy3, Cy5, Cy5.5, Daboyl, digoxigenin, dinitrophenyl, Edans, 6-FAM, fluorescein, 3'glyceryl, HEX, IRD-700, IRD-800 , JOE, psoralen phosphate, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S, SE, BODIPY®, Marina Blue®, Pacific Blue®, Oregon Green®, Rhodamine Green®, Rhodamine Red®, Rhodol Green® and Texas Red®. Preferably, said additions are incorporated at the 3 'or 5' end of a nucleic acid sequence used as RT and / or RTDD and / or interaction domain as part of the artificial molecular complex of the present invention. This modification has the advantageous effects that the cellular location of the RTDD and / or the interaction domain and / or the DNA repair model nucleic acid sequence within a cell can be visualized to study the distribution, concentration and / or availability of the respective sequence. In addition, the interaction of the artificial molecular complex of the present invention with endogenous molecules can be studied. Methods for studying similar interactions or for viewing a modified or labeled nucleotide sequence as detailed above are available to the person skilled in the art in the respective field.
[00176] For any embodiment according to the various aspects of the present invention, the at least one site-specific nuclease and / or at least one repair model nucleic acid sequence and / or the at least one interaction domain and / or the at least one RTDD comprises at least one nuclear localization sequence (NLS), a plastid localization sequence (PLS), preferably a mitochondrial localization sequence or a chloroplast localization sequence. Therefore, at least one of the components of the artificial molecular complex comprises a sequence to direct the complex towards the
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117/202 nuclear genome. In certain embodiments, RTDD can also load at least one location string. Preferably, the SSN and / or the interaction domain of the artificial molecular complex will comprise at least one NLS or at least one PLS, or both will comprise at least one NLS and at least one PLS sequence. This at least one sequence of NLS or PLS will transport the entire artificial molecular complex to the nucleus. Proteins labeled with NLS or PLS can be generated as fusion molecules labeled with NLS or PLS.
[00177] For modalities, in which the artificial molecular complex according to the present invention is used for in vitro purposes, for example, to modify a genome or part of a genome, on a plasmid or any other vector in vitro, it may not no location sequence is required. The localization sequences help to direct the artificial molecular complex to at least one target DNA sequence of interest in the relevant compartment within a target cell of interest. According to certain embodiments of the present invention, the localization sequence can comprise a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence or a chloroplast localization sequence. Therefore, the at least one SSN and / or at least one RTDD and / or at least one interaction domain will comprise at least one corresponding location sequence, preferably a nuclear location sequence (NLS) to target the complex to the nuclear genome of the cell. In some embodiments, the SSN and / or the RT and / or the at least one interaction domain and / or the RTDD may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, or more NLSs at or near the amino terminus (for peptides and proteins), about or
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118/202 more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy terminus (for peptides and proteins), or a combination of these (for example , one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus for peptides and proteins). Components of the artificial molecular complex not based on amino acids will carry the localization sequence, for example, on the 5 'and / or 3' end, as is the case for nucleic acid sequences. In addition, a localization sequence, preferably a synthetic localization sequence, can also be added at any position within a molecule as long as it does not disturb the interactions within the molecular complex and / or the capacity for binding, dividing and repair of the artificial molecular complexes of the present invention. When more than one NLS is present, each can be selected independently of the others, such that a single NLS can be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies.
[00178] In a preferred embodiment of the invention, the at least one SSN and / or the interaction domain will comprise a localization sequence and can comprise a maximum of 6 NLSs. In some embodiments, an NLS is considered close to the amino termination or the carboxy terminus of an amino acid component of the artificial molecular complex when the amino acid closest to the NLS is within about 1, 2, 3, 4, 5, 10, 15 , 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain of the amino terminus or the amino carboxy terminus. Non-limiting examples of NLSs include an NLS sequence derived: from the SVL virus large T antigen NLS, having the amino acid sequence PKKKRKV (SEQ ID NO: 1); nucleoplasmin NLS (for example, the nucleoplasmin bipartite NLS with the sequence
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119/202
KRPAATKKAGQAKKKK (SEQ ID NO: 2)); NLS c-myc having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or
RQRRNELKRSP (SEQ ID NO: 4); of the NLS hRNPAI M9 having the sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); of the sequence
RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) from domain 188 of importin-alpha; the VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) sequences of the myoma T protein; the PXPKKKPL sequence (SEQ ID NO: 9) from human p53, where the L at position 8 of SEQ ID NO: 9 is optional; mouse SALIKKKKKMAP (SEQ ID NO: 10) sequence of c-abl IV; the DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) sequences of the influenza virus NS1; the RKLKKKIKKL sequence of the Hepatitis virus delta antigen (SEQ ID NO: 13); the REKKKFLKRR sequence (SEQ ID NO: 14) of the mouse Mx1 protein; the KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) sequence of human poly (ADP-ribose) polymerase; and the RKCLQAGMNLEARKTKK (SEQ ID NO: 16) sequence of the steroid hormone receptor (human) glucocorticoid. In some embodiments, the location signal can be a plastid location signal, for example, a plastid location signal or a mitochondrial location signal. Suitable plastid localization signals are selected from the group consisting of chloroplast transit peptides or mitochondrial targeting peptides. In addition, peptides derived from the HIV Tat protein, or sequences encoding it, may be suitable for targeting a construct or molecule of interest into a cell and / or a subcellular compartment of interest. Suitable Tat peptides are derived from YGRKKRRQRRR (SEQ ID NO: 17)
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120/202 or comprise the GRKKR motif (SEQ ID NO: 18). In another example embodiment, a sequence derived from yeast mitochondrial Cox4p (SEQ ID NO: 30) or a sequence derived from the mitochondrial leader sequence of human malate dehydrogenase (MLS) (SEQ ID NO: 31) or derived from lipoic acid synthase of Arabidopsis (NCBI Ref. Seq. ID: NP 179682.1 designated herein, in this patent application, as SEQ ID NO: 32) can be used to locate the artificial molecular complex according to the present invention into a mitochondrial matrix so to modify mitochondrial DNA.
[00179] In particular modalities, it may be of interest to direct the artificial molecular complex to the chloroplast. In many cases, this targeting can be accomplished by the presence of an N-terminal extension, called a chloroplast transit peptide (CTP) or plastid transit peptide. Chromosomal transgenes from bacterial sources must have a sequence encoding a CTP sequence fused to a sequence encoding an expressed polypeptide if the expressed polypeptide is to be compartmentalized in the plant's plastid (eg chloroplast). Therefore, localization of an exogenous polypeptide to a chloroplast is often accomplished by operationally linking a polynucleotide sequence encoding a CTP sequence to the 5 'region of a polynucleotide encoding the exogenous polypeptide, that is, at least one SSN according to the present invention. The CTP is removed at a processing step during translocation into the plastid. However, processing efficiency can be affected by the amino acid sequence of the CTP and by close sequences at the NH2 terminus of the peptide. Other options for targeting to the chloroplast which have been described are the signal sequence cabPetição 870190072502, of 07/29/2019, pg. 133/367
121/202 m7 of corn (U.S. Patent No. 7,022,896, WO 97/41228) a pea glutathione reductase signal sequence (WO 97/41228) and the CTP described in United States patent application No.
US 2009/029861 A1.
[00180] The various localization sequences according to the present invention can be encoded on an expression plasmid or cassette encoding at least one localization sequence to operationally link the localization sequence to the respective molecule, or the localization sequences can be attached to a protein, nucleic acid or other biomolecule forming the artificial molecular complexes of the present invention in a synthetic way.
[00181] In yet an additional modality, at least one nuclear export signal can be used in addition to or instead of at least one location sequence.
[00182] In modalities, in which the artificial molecular complex is released to a cell with the aid of at least one release vector in the form of a nucleic acid sequence, the localization signal can be covalently attached to at least an SSN and / or the sequence encoding the interaction domain in a covalent mode as a nucleic acid sequence encoding a localization signal.
[00183] In one embodiment, at least one SSN and / or a polypeptide interaction domain can be associated covalently or non-covalently with a fluorescent reporter gene or protein. This reporter can be released as DNA, as mRNA, as an independent protein, or as a fusion protein linked to at least one SSN and / or the polypeptide of the interaction domain.
[00184] The RTDD / RT molecule according to the present
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The invention can be produced in several ways. It can be produced by chemical synthesis, adding RNA bases where appropriate in the synthesis process and DNA bases where appropriate in the synthesis process. Alternatively, RTDD and / or RT can be synthesized independently of each other and the molecules can then be associated with each other as described above. Another option is to use T4 RNA ligase or another enzyme capable of binding nucleic acids to RNA, preferably single-stranded RNA. Here, the RNA and DNA components are generated independently by any method, mixed, exposed to the enzyme according to the manufacturer's protocol, and will be covalently linked by bonding, that is, to generate covalent fixation. Other strategies for covalent attachment of RTDD to RT include the attachment of each to other chemical binding groups or complexes, such as a peptide. This type of approach is especially suitable when the hybrid RTDD / RT sequence must be detected later within the cell, or when an additional function must be assigned to the hybrid nucleic acid sequence. Chemical modification of either the RTDD nucleic acid sequence and / or the RT nucleic acid sequence can be of great importance to stabilize the RTDD / RT sequence and to prevent degradation by cellular enzymes in order to obtain simultaneous high availability of the sequence RTDD / RT and at least one site-specific nuclease at the target DNA site of interest.
[00185] In embodiments, where RTDD is a gRNA and SSN is a CRISPR nuclease, preferably a Cas or Cpf1 nuclease, more than one RTDD may be present. It has been seen that using multiple gRNAs simultaneously will increase CRISPR-based gene activation or repression and can significantly reduce the emergence of conduction-resistant alleles
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123/202 genetic. Therefore, gRNAs as RTDDs can be presented as a single unprocessed transcript and the gRNAs will then be excised from the precursor in the nucleus by RNA polymerase II transcription simultaneously preventing the export of gRNA to the cytoplasm (Port and Bullock, Nat. Methods, 2016 , vol. 13, no.10, 852854). In these modalities, gRNAs can be presented as tRNA-gRNA plasmids so that the endogenous tRNA processing mechanism will release multiple functional gRNAs.
[00186] According to certain modalities of the various aspects of the present invention, the at least one site-specific nuclease, or the sequence encoding the same, and the at least one interaction domain, or the sequence encoding the same, and / or the hair least one repair model docking domain, or the sequence encoding it, are connected by at least one linking domain. This linker sequence can serve as a molecular spacer to obtain optimal geometry of the RTDD sequence and the repair model nucleic acid sequence as well as the SSN and optionally the interaction domain component of the artificial molecular complex so that all individual components fully exercise their function. The length and composition of the binder or cable (tether) regions can be an important design aspect, for example, for some RTDD and RT pairs. In one embodiment, especially the 5 'end of the left homology arm of the RT may comprise a ligand region. The cable or binder can take a variety of shapes. Starting from the left or right homology arm of the RT, allowing this portion of the RT to act as a cable or as a flexible ligand to enable movement of the RT towards the chromosomal target, and once homology
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124/202 to mediate the HR reaction can be performed by the person skilled in the art based on the present disclosure and having knowledge of the usual design parameters for repair models as currently used widely for genome editing.
[00187] In embodiments, in which the artificial molecular complex comprises at least one SSN as well as at least one interaction domain (AI), the SSN and the AI can be connected by a suitable ligand.
[00188] Design parameters to be considered include the geometry of the homology of the repair model in relation to the cut site of a CRISPR polypeptide such as SSN, the chain within a target DNA site of interest to which the repair model is homologous , the size of the repair model, which can influence whether a binder and at what length a binder will be introduced. A ligand sequence can be used for both covalent and non-covalent associations of the gRNA and the repair model. Based on the present disclosure and based on the information provided in Nishimasu (supra), Tsai et al. (Nature Biotechnology, 32, 569-576, (2014)), or Shechner et al. (Nature Methods, 12 (7), 664-670 (2015), doi: 10.1038 / nmeth.3433), the person skilled in the art can therefore define a suitable ligand region for a hybrid nucleic acid sequence to define a sequence specific between the gRNA as RTDD and the RT or between different gRNAs and or RTs, if several hybrid nucleic acids are used so that both the gRNA and the RT portion can fully perform their function without any steric restrictions. The at least one linker region can comprise up to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 additional nucleotides for properly separate at least one gRNA from the RT, or to optimize the positioning of the gRNA and / or the RT. In certain
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125/202 modalities, the ligand sequence can comprise up to 150, 200,
250, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or at least 4,700 or 5,000 nucleotides to achieve better positioning of the gRNA and / or RT.
[00189] For non-covalent association of any nucleic acid sequences comprising RTDD with RT, an approach is to provide partially complementary sequences in RTDD and RT so that the two molecules will naturally associate by nucleic acid base pairing.
[00190] Other methods of non-covalent association are conceivable, such as the use of electrical charges of molecules to cause a sufficient association of RT with some component of the artificial molecular editing complex. In another embodiment, at least one component of the artificial molecular complex, may comprise a marker and the binding partner, i.e., RTDD and SSN, or RTDD and the interaction domain, and / or the RT portion of it , respectively, comprise the corresponding binding partner of the marker so that a non-covalent interaction, optionally in addition to the base pairing between RTDD or the interaction domain and RT and the association between RTDD and the SSN polypeptide is performed to increase the interaction and therefore the stability of the artificial molecular complex.
[00191] In human cells, Cas9 loaded with a gRNA having an additional 28 bp sequence on the 3 'end plus an associated 187 amino acid (21.4 kD) Csy4 protein maintained at least 90% activity in chain break induction double compared to routine gRNA controls (Tsai et al., Nature Biotech., 32, 2014). This suggests a very substantial tolerance for Cas9 as SSN for charge tied to the 3 'end of the sgRNA and for potential structure function suitable for the molecule of
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126/202 extended sgRNA. The tolerance of Cas9 is made possible in part by the flexibility of the free 3 'end of the nucleic acid sequence, which in a standard gRNA ends in a hairpin that is kept outside the Cas9 protein architecture and on a surface roughly perpendicular to the enclosing surface the active site (Nishimasu et al., 2014; Anders et al., 2014). In addition, Shechner et al. (Multiplexable, locus-specific targeting of long RNAs with CRISPRDisplay, Nature Methods, 12 (7), 664-670 (2015), doi: 10.1038 / nmeth.3433) show that long non-coding ssRNA molecules can be attached transcriptionally at the 5 'or 3' ends of the sgRNA, or in an inner loop of the sgRNA without loss of sequence-specific targeting activity by a dCas9 protein in the human cell genome. ssRNAs up to 4.8 kb were accommodated by the ribonucleoprotein complex with maintenance of the specific targeting activity for the sequence.
[00192] In an embodiment according to the various aspects of the present invention, the repair model nucleic acid sequence is associated with RTDD, for example, a guide nucleic acid sequence, at the 3 'end of the nucleic acid sequence guide, and / or the repair model nucleic acid sequence is associated with the 5 'end of the RTDD, for example, a guide nucleic acid sequence, and / or the repair model nucleic acid sequence is located within the RTDD and thus forms a separate functional part of RTDD.
[00193] Surprisingly, it was seen by the inventors of the present invention that a gRNA like RTDD carrying a localized 3 'DNA repair model sequence (RT), either a single-stranded RT or a double-stranded RT, was free to interact with homologous sequence as it is released to the target by a
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127/202 CRISPR polypeptide, for example, Cas9 from a CRISPR Type II system, or Cpf1 from a CRISPR Type V system or other effector CRISPR polypeptide. Similar observations were made when a gRNA was used as RTDD carrying the 5 'localized DNA repair model sequence, or a single strand RT or a strand RT, or both, a 3' and 5 'localized RT. Located 3 'or 5' therefore implies that the RT is either covalently attached to the 3 'or 5' end of a gRNA, or it may mean that the RT is hybridized to, that is, non-covalently associated with, a region corresponding to the sequence attached to the 3 'and / or 5' region of the gRNA. In addition, the RT can be covalently incorporated into the stem loops of a gRNA, or it can be non-covalently associated with said gRNA stem loops to obtain a functional hybrid nucleic acid construct, wherein the RTDD and the RT are interacting directly. Thus, it was seen that DNA associated with a gRNA in various positions of the gRNA as described above was well tolerated and this new form of hybrid complex is therefore suitable to bring together two key aspects of the principle of genetic editing: (1) precision of targeting mediated by RTDD / gRNA and (2) efficient and site-directed repair as mediated by RT. In addition, there is a synergistic effect that gRNA and RT are brought in close proximity in order to increase the stability and availability of the hybrid construct together with a CRISPR polypeptide as an SSN of interest at a target DNA site of interest.
[00194] There are almost no limitations on the length of this extended repair model nucleotide sequence released as part of the artificial molecular complex according to the present invention, in case the RT is attached to the 3 'or 5' end of a Nucleic acid based RTDD, for example,
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128/202 is a gRNA or a gDNA. The length of the RT, regardless of the type of RTDD, is preferably dictated by the targeted modification to be introduced. Typical RT sequences can have a length from about 20 to 8,000 bp or even more, for example, from 20 to 5,000 bp, from 30 to 8,000 bp, from 30 to 5,000 bp, from 40 to 8,000 bp, from 40 at 5,000 bp, from 50 to 8,000 bp, from 50 to 5,000 bp, from 60 to 8,000 bp, from 60 to 5,000 bp, from 70 to 8,000 bp, from 70 to 5,000 bp, from 80 to 8,000 bp, from 80 to 5,000 bp, from 90 to 8,000 bp, from 90 to 5,000 bp, from 100 to 8,000 bp, from 100 to 5,000 bp of single-stranded and / or double-stranded DNA without a significant loss in the cutoff frequency of at least one SSN. As is known to the person skilled in the art, the length of a RT model is strongly dictated by the type of modification / insertion to be made / introduced. In the event that a knock-in of a larger nucleic acid sequence encoding a protein of interest is desired, the length of the RT sequence will have the length: length of the nucleic acid construct encoding the protein of interest plus two arms of sufficient homology long ones located to the left and right of the sequence. Thus, there is in principle no upper limit of 1,500 bp, but the RT can have up to 5,000 or even more base pairs (bp). For example, larger inserts presently introduced using plasmid DNA as a repair model and producing the repair model within a target site use 800 bp left and right homology arms and more so that the total length of a model repair may have several 1,000 bp. The length of the nucleic acid inserts will be designed to not inhibit the site-specific nuclease of interest which can be determined in pre-experiments.
[00195] The different components of the molecular complex of the present invention, that is, at least one SSN, the at least one
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RTDD and at least one RT, and optionally at least one interaction domain are associated in a functional mode.
[00196] The term associated in a functional mode implies that the components of the artificial complex are brought into contact so that the SSN and the RTDD can interact with each other, preferentially by a form of non-covalent association, as detailed above . The at least one RTDD sequence interacting with at least one RT sequence are mounted independently, either before, after, or simultaneously with the contact of at least the RTDD sequence with at least one corresponding SSN, or a variant or a catalytically active fragment of the same interest. In one embodiment, the entire complex, optionally comprising at least one interaction domain, is associated in vitro before being introduced into a target cell comprising at least one target DNA region of interest to be edited. In another embodiment, at least one SSN and optionally an interaction domain are introduced into at least one target cell before or after at least one interaction RTDD / RT sequence. The SSN polypeptide can be introduced into a target cell by transfecting the polypeptide sequence or by transfecting or transforming at least one target cell with RNA encoding the at least one SSN polypeptide or by introducing a release construct encoding at least one SSN polypeptide which can be transcribed and translated into a target cell. Likewise, in certain embodiments, one or more RTDD sequences and one or more repair model nucleic acid sequences can be provided simultaneously as provided in vitro and the assembled construct. Alternatively, either the RTDD sequence and / or the repair model nucleic acid sequence can
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130/202 be transfected or transformed within a target cell with the aid of a suitable release vector. In a preferred embodiment, the entire artificial molecular complex is assembled in vitro and then introduced into a target cell of interest to allow better spatial and stoichiometric control of the genome editing construct. In another preferred embodiment, at least one SSN and optionally an interaction domain polypeptide is introduced into a target cell before the RTDD / RT sequences and at least one RTDD / RT sequence is then introduced into a target cell of interest after that. Sequential order may be preferable for some approaches using, for example, a gRNA as RTDD due to the intrinsically low RNA stability compared to a polypeptide, so that the introduced gRNA will be immediately bound and stabilized by the SSN, that is, for certain modalities a CRISPR polypeptide already present in the cell. Without wishing to be bound by theory, the ex vivo assembly of a guide nucleic acid sequence and a repair model nucleic acid sequence can also enhance the stability of the construct compared to the isolated guide RNA.
[00197] Currently, there are a variety of methods of transforming plants to introduce genetic material in the form of a genetic construct into a plant cell of interest, comprising biological and physical means of knowledge of the expert in the plant biotechnology technique. A common biological medium is transformation with Agrobacterium spp., Which has been used for decades for a variety of different plant materials. The transformation of plants mediated by viral vectors represents an additional strategy for the introduction of genetic material into a cell of interest. Physical means finding application in
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131/202 plant biology is particle bombardment, also called biolistic transfection or microparticle-mediated gene transfer, which refers to a mathematical release method for the transfer of a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest within a target cell or tissue. The physical introduction means are suitable for introducing nucleic acids, i.e., RNA and / or DNA, and proteins. Likewise, specific transformation or transfection methods exist to specifically introduce a nucleic acid or an amino acid construct of interest into a plant cell, including electroporation, microinjection, nanoparticles, and cell-penetrating peptides (CPPs). In addition, chemical-based transfection methods exist to introduce genetic constructs and / or nucleic acids and / or proteins, comprising, among others, transfection with calcium phosphate, transfection using liposomes, for example, cationic liposomes, or transfection with cationic polymers , including DEAD-dextran or polyethyleneimine, or combinations thereof. Said delivery methods and delivery vehicles or charges therefore inherently differ from delivery tools as used for other eukaryotic cells, including animal and mammalian cells, and each delivery method needs to be specifically adjusted and optimized in order for a construct of interest to mediate genome editing can be introduced into a specific compartment of a target cell of interest in a fully functional and active way. The above release techniques, alone or in combination, can be used to insert at least one artificial molecular complex, or at least one subcomponent thereof, that is, at least one SSN, at least one RTDD, at least one RT and optionally at least one
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IA, or the sequences encoding the aforementioned subcomponents, according to the present invention within a target cell, in vivo or in vitro.
[00198] In certain embodiments, modes of release of the artificial molecular complex of the present invention can be selected from PEG-mediated release of an SSN- (IA) -RTDD-RT complex, PEG-mediated release of plasmid encoding SSN- (IA ) -RTDD, RTDD for example, being a gRNA or gDNA and parallel release of RT, bombardment of a complex of SSN- (IA) -RTDD-RT, bombardment of plasmid encoding protein (SSN and optionally IA) -RTDD, by example, gRNA / gDNA, and parallel RT release, cell-penetrating peptide-mediated (CPP) release of an SSN- (IA) RTDD-RT complex, lipofection of an SSN- (IA) -RTDD-RT complex, plasmid lipofection encoding protein (SSN and optionally IA) -RTDD, for example, gRNA / gDNA, and parallel release of RT, or stable expression of protein (SSN and optionally IA) and transient release of RTDD or, for certain RTDDs, a plasmid encoding RTDD and parallel release of rtDNA.
[00199] In certain embodiments, the crRNA portion of the gRNA comprises a stem loop or an optimized stem loop structure or an optimized secondary structure. In another embodiment, the mature crRNA comprises a stem loop or stem loop structure optimized in a direct repeat sequence, in which the stem loop or the stem stem structure optimized is important for diving activity. In certain embodiments, the mature crRNA preferably comprises a single stem loop. In certain embodiments, the direct repeat sequence preferably comprises a single stem loop. In certain modalities, the divage activity of the
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The effector protein complex is modified by introducing mutations that affect the structure of the stem loop RNA duplex. In preferred modalities, mutations can be introduced which maintain the RNA duplex of the stem loop, through which the dividing activity of the effector protein complex is maintained. In other preferred embodiments, mutations can be introduced which disrupt the structure of the RNA duplex of the stem loop, through which the dividing activity of the effector protein complex is completely abolished.
The size of the at least one repair model nucleic acid sequence according to the present invention as part of the artificial molecular complex according to the present invention can vary. It can be within a range of about 20 bp to about 5,000 bp or even 8,000 bp depending on the target DNA sequence to be modified.
[00201] HOR models used to create specific mutations or insertions within a target DNA region of interest require a certain amount of homology around the target sequence that will be modified. It is best if the insertion sites of the modification are no more than 100 bp away from the double strand break (DSB) as performed by an SSN or a fusion partner, that is, a domain of interaction, in the case of a Nuclease-deficient SSN, for example, a CRISPR polypeptide, ideally less than 10 bp apart if possible, and the total length of the homology arm is an important factor to consider when designing these. Longer distances will work, but efficiency is likely to be less and the introduction of a selection marker may become necessary to ensure that the desired modification to be introduced into the target DNA sequence of interest is present.
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According to the various aspects of the present invention, the at least one repair template nucleic acid sequence can be a single-stranded or double-stranded DNA nucleic acid molecule. The at least one repair model nucleic acid sequence can be provided in the form of one or more linear DNA molecules, ss- or ds-DNA. However, it may be appropriate to use at least one single-stranded or double-stranded repair model nucleic acid sequence, which is produced ex vivo, when a molecular complex must be assembled ex vivo, which is especially suitable for increasing the availability of the functional SSN-RTDD-RT complex, since all components can be introduced simultaneously in the correct stoichiometry to increase the specificity of the genome editing approach.
[00203] The synthesis of larger nucleic acid sequences, either single-stranded or double-stranded, can be performed using common methods of the prior art. It should be noted that for certain embodiments, partial single strand and / or partial double strand repair nucleic acid sequences may also be suitable. Any combination of a single-stranded and / or double-stranded nucleic acid sequence and any type of introduction is possible, whether simultaneous with or before or after the introduction of the polypeptide components of the artificial molecular complex. In one embodiment, it is envisaged to introduce a molecular complex according to the second aspect into a target cell, where the target cell comprises an additional plasmid vector encoding a repair model or an additional repair model sequence, since the use of more than one repair model nucleic acid sequence is beneficial for certain genome editing approaches, where the complex
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The artificial molecular 135/202 can then assemble in vivo after the different components are provided. In general, the high physical availability of the repair model nucleic acid sequence at that site within a target cell, where the target DNA region is located is of utmost importance to allow for a highly accurate genome editing event. In certain modalities, single-stranded DNA (SS) repair models are especially suitable to strike the correct balance, keeping the molecular weight as low as possible, while providing sufficient length for homology interactions, in order to achieve optimal homology-directed repair.
[00204] In an embodiment according to any aspect of the present invention, the at least one SSN is a CRISPR polypeptide and is independently selected from the group consisting of a Cas polypeptide from Streptococcus spp., Including Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria spp., Including Neisseria meningitides, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azosvacobacteria, Rosaceae Candidatus Micrarchaeum acidiphilum ARMAN-1, Parcubacteria (GenBank: APG80656.1), Sulfolobus spp., Including Sulfolobus islandicus HVE10 / 4 (GenBank: ADX81770.1) or REY15A (GenBank: ADX84852.1), or where the CRISPR polypeptide is selected from a Cpf 1 polypeptide from an archaea or a bacterium, including a Cpf1 polypeptide from Acidaminococcus spp., including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., Including Lachnospiraceae bacterium ND2006, Francisella spp., Including Francisella novicida U112, Eubacterium eligens, Prevotella spp., Or
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Porphyromonas spp., Or variants and / or functional fragments and / or combinations thereof, including polypeptide nickases
CRISPR, or a CRISPR polypeptide lacking endonucleolytic activity.
[00205] In an embodiment according to the present invention, the RTDD / RT sequences according to the present invention can be used with an SSN nickase, for example, a Cas9 nickase, mutant to minimize off-target mutations, in which they are paired guide RNAs are used, each of which is specific for a Cas9-derived nickase mutant.
[00206] In some embodiments, at least one SSN and optionally the at least one interaction domain is provided as an expressed, translated or synthesized polypeptide in vitro. In some embodiments, a release vector encoding at least one CRISPR polypeptide is used, wherein the release vector may additionally comprise regulatory sequences or localization signals. A SSN polypeptide that is mutated with respect to a corresponding wild-type enzyme such that the mutated SSN enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence also comprised of various embodiments in accordance with the present disclosure. For example, an aspartate-by-alanine (D10A) substitution can be used in the catalytic RuvC I domain of Cas9 from S. pyogenes which converts Cas9 from an endonuclease that cleaves both strands of a target DNA region of interest to a nickase cleaving a single chain. Other examples of mutations that make a Cas9 polypeptide a nickase include, without limitation, H840A, N854A, and N863A. As an additional example, two or more catalytic Cas9 domains (RuvC I, RuvC II, and RuvC III or the HNH domain) can be mutated to produce a mutated Cas9
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137/202 substantially lacking any DNA dividing activity. In some embodiments, a D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA dividing activity. In some embodiments, an SSN enzyme is considered to lack substantially all of the DNA dividing activity when the DNA dividing activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0, 1%, 0.01%, or less of the DNA dividing activity of the unmuted form of the enzyme; an example may be when the DNA dividing activity of the mutated form is null or negligible as compared to the unmutated wild form. When the enzyme is not Cas9 from S. pyogenes, mutations can be made to any or all of the residues corresponding to positions 10, 762, 840, 854, 863 and / or 986 of SpCas9 (which can be determined, for example , by routine sequence comparison tools). In particular, any or all of the following mutations in Cas9 of S. pyogenes are preferred: D10A, E762A, H840A, N854A, N863A and / or D986A; as well as a conservative substitution for any of the replacement amino acids according to the present disclosure. The same conservative substitutions or substitutions for these mutations at corresponding positions in other Cas9s are also possible for certain modalities, particularly D10 and H840 in S. pyogenes Cas9. However, in other Cas9s, residues corresponding to D10 and H840 Cas9 from S. pyogenes are also possible. Orthologists or orthologists of certain CRISPR proteins can also be used in the practice of the invention. Orthologists are genes in different species that evolved from a common ancestral gene through speciation. Typically, orthologists retain the same function in the course of
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138/202 evolution. Most preferably, the Cas9 enzyme as SSN is from, or is derived from, Cas9 from S. pyogenes, or Cas9 from S. aureus, or wild Cas9 from S. thermophilus, whose protein sequence is given in the database SwissProt under access number G3ECR1. Similarly, Cas9 from S. pyogenes or Cas9 from S. aureus is included in the SwissProt under accession number Q99ZW2.
[00207] In one embodiment, the guide RNA as RTDD sequence according to the present invention is designed to have optimal activity, that is, recognition properties, for a selected SSN enzyme or polypeptide of a specific length, the SSN enzyme can therefore be truncated to a catalytically active fragment of the wild SSN making it shorter in length than the corresponding wild enzyme by truncating the nucleic acid molecules encoding the SSN enzyme which can be transcribed or translated in vitro or in vivo, or providing a synthesized SSN polypeptide. It is also possible to produce chimeric SSN enzymes, in which different parts of the enzyme are exchanged or exchanged between different orthologists to arrive at chimeric enzymes having adapted specificity.
[00208] A functional variant or fragment according to the present disclosure therefore comprises any SSN protein and / or interaction domain and / or RTDD or a truncated version of the same derived from the SSN protein and / or interaction domain and / or Wild RTDD, that is, having a degree of sequence homology with, a wild-type enzyme, but which has been mutated (modified) in some way as described herein, in this patent application. For example, the nuclease enzymatic activity derived from Cas9 generates double strand breaks in target site sequences which hybridize to 20 nucleotides of the guide sequence and which have a motif sequence adjacent to - (PAM), examples including
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NGG / NRG or a PAM that can be determined as described here, in this patent application, following the 20 nucleotides of the target sequence. This enzymatic function can be varied by generating variants of SSN having nickase activity or dead variant of nuclease. In addition, a variant of the SSN polypeptide and / or interaction domain and / or RTDD according to the present disclosure can be codon-optimized to adapt the SSN polypeptide and / or interaction domain and / or RTDD to the use of codon from a target cell, preferably a eukaryotic cell, preferably an animal cell or a plant cell.
[00209] In preferred embodiments according to the present invention, the components of the artificial molecular complex, particularly the components of SSN or IA, or the catalytically active fragments thereof still exercising the catalytic function of the wild polypeptide, and / or the additional components can be codon optimized, and / or the SSN polypeptide and / or the interaction domain, and / or RTDD and / or RT can be linked to a marker sequence, in order to identify the location of a target sequence and / or the artificial molecular complex. The marker can be selected from the group consisting of a polyhistidine (His) -Tag marker, a glutathione-S-transferase (GST) marker, a thioredoxin marker, a FLAG marker, a marker having fluorescent properties, for example, selected from (E) GFP marker ((enhanced) green fluorescent protein), a DsRed-tag, an mCherry-tag, a (t) dtomato-tag, an mNeonGreen-tag and the like or, a streptavidin or strep-tag, a maltose-binding protein (MBP) marker, a transit peptide allowing targeting to a subcellular compartment, including mitochondria or the nucleus, a snap-tag and / or a secretion marker allowing the secretion of an amino acid sequence
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140/202 attached to it, an unnatural amino acid that does not normally occur in nature, or a combination of the aforementioned markers. A protein component of the artificial molecular complex, for example, the SSN and / or the interaction domain, can comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to any component of at least one artificial molecular complex include, without limitation, epitope markers, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, activity demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA dividing activity and nucleic acid binding activity. Non-limiting examples of induce epitope markers, histidine markers (His), V5 markers, FLAG markers, influenza hemagglutinin (HA) markers, Myc markers, VSV-G markers, and thioredoxin markers (Trx). Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), strong root peroxidase (HRP), doranphenicol acetyltransferase (CAT) beta-galactosidase, betaglucuronidase, luciferase, green fluorescent protein (GFP), HcRed , DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme can be fused to a gene sequence encoding a protein or fragment of a protein that binds DNA molecules or binds other cell molecules, including, but not limited to, maltose binding protein (MBP), S-tag , Lex A fusions to the DNA binding domain (DBD), GAL4 fusions to the DNA binding domain, and virus fusions
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141/202 herpes simplex (HSV) protein BP16.
[00210] In one embodiment, at least one component of the artificial molecular complex may be a modified one functioning as DNA nickase, and / or the SSN polypeptide, or the catalytically active fragment thereof, may be present in the form of a fusion molecule with another functional portion, preferably a functional polypeptide portion having enzymatic function, preferably a functional portion having chromatin modeling function, and / or stimulating homologous recombination, and / or transcription modification. When analyzing at least one modified cell within a tissue of a multicellular organism, said markers and marker proteins are preferred, especially fluorescent protein markers, which have a bright fluorescence, so that they can be determined even in deeper layers of complex tissues. Suitable fluorescent proteins are commercially available and can be easily selected for the specific purpose by the person skilled in the art.
[00211] According to the various embodiments of the present invention, either the SSN polypeptide (s) and / or interaction domain and / or RTDD and / or the RTDD sequence and / or RTs comprise at least one nuclear localization sequence, and / or a plastid localization sequence, for example, a mitochondrial localization sequence or a chloroplast localization sequence, for efficient targeting of the SSN polypeptide to a cell compartment comprising a sequence of Genomic DNA of interest to be modified. The sequence requirements for said location sequences are known to the person skilled in the art of molecular biology. In order not to hinder the function of the SSN polypeptide or the nucleotide sequence of RT, the
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142/202 localization sequence is fused, that is, covalently linked, to the amino termination or the carboxy terminus, or correspondingly to the 5 'or 3' end of the respective molecule. [00212] In one embodiment, the at least one SSN polypeptide and optionally the at least one interaction domain, if representing a polypeptide sequence, are provided as a polypeptide sequence produced ex vivo, either using recombinant technologies for protein production or by synthesizing the corresponding amino acid sequence. In another embodiment, the SSN polypeptide and optionally at least one interaction domain is presented as an RNA sequence, which can be translated into the corresponding amino acid sequence after the introduction of a target cell of interest. In yet an additional embodiment, the SSN polypeptide and optionally the at least one polypeptide interaction domain are inserted as a DNA construct, either configured for stable expression or for transient expression in a cell of interest, so that the at least one polypeptide of SSN and optionally the at least one polypeptide interaction domain are then transcribed and translated into a target cell of interest in a constitutive or inducible manner. Suitable DNA constructs and associated methods for introducing at least one SSN polypeptide and optionally at least one polypeptide interaction domain according to the present invention into a target cell are known to the person skilled in the art, whereas modes Specifics for introducing the at least one SSN polypeptide and optionally the at least one polypeptide interaction domain according to certain embodiments of the present invention specifically adapted for application to plant cells are further detailed below.
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143/202 [00213] The artificial molecular complex, or parts thereof, that is, at least one SSN polypeptide, at least one RTDD and at least one RT and optionally at least one interaction domain have been introduced within a target cell of interest using an appropriate release construct. Naturally, the type of release construct can vary, depending on whether the molecular complex is fully assembled in vitro and subsequently introduced into a target cell, or whether the different components of the molecular complex are introduced separately into a cell and the complex is then assembled by non-covalent interactions within a target cell of interest. Introduction usually takes place using a suitable release construct.
[00214] The term release construct or vector (of release) as used here, in this patent application, according to various modalities of the different aspects of the present invention refers to any biological or chemical means, or non-chemical or based on particles and / or methods used as a charge for transporting a nucleotide sequence and / or an amino acid sequence of interest into a target eukaryotic cell. Release constructs comprise biological means for the release of nucleotide sequences within a target cell, including viral vectors, Agrobacterium spp., Cell penetrating peptides (CPPs) or chemical release constructs, including nanoparticles, lipid or polymeric vesicles, phosphate from calcium, or combinations thereof. Lipid or polymeric vesicles can be selected, for example, from lipids, liposomes, lipid encapsulation systems, nanoparticles, for example, mesoporous silica nanoparticles, formulations of small particles of nucleic acid-lipids, polymers, for example, cationic polymers such as
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144/202
DEAE-dextran or polyethyleneimine and polymersomes. In one embodiment, the polymer is selected from the group consisting of linear polymers, branched polymers, dendrimers (highly branched organic compounds), and polysaccharides. In another embodiment, the lipid encapsulation system comprises one or more of a phospholipid, cholesterol, polyethylene glycol (PEG) -lipid, and a lipophilic compound that releases the particle into the target tissue. In an additional embodiment, the release construct can be a mesoporous silica nanoparticle.
[00215] Methods of physical introduction as used herein, in this patent application, and as appropriate to provide at least one molecular complex or at least one hybrid RNA / DNA nucleic acid sequence according to the present invention refer to electroporation, microinjection, particle bombardment, sonoporation, magnetofection or impalefection using elongated nanostructures and matrices of similar nanostructures such as carbon nanofibers or silicon nanowires which have been functionalized with plasmid DNA, and chemical methods and can be based on the use of micro- or nanoparticles or chemicals, including polyethylene glycol (PEG).
[00216] For example, for a modality, where the components of the artificial molecular complex are associated ex vivo, the release vector can be a lipid-based vector or a polymeric vector. Vectors based on lipids or polymers can be selected, for example, from lipids, liposomes, encapsulation systems of lilpids, microparticles, whiskers, nanoparticles, small particles of nucleic acid-lipids, polymers, and polymersmas. In some embodiments, the polymer can be selected from the group consisting of linear polymers, branched polymers, dendrimers, and polysaccharides. In another modality, the
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145/202 encapsulation of lilpids comprises one or more of a phospholipid, cholesterol, polyethylene glycol (PEG) -lipid, and some lipophilic compounds that release the particle into a target cell.
[00217] For mammalian cells, ex vivo modification of immune cells for various therapeutic purposes has gained much interest during the last decade to combat various tumor diseases by adoptively transferring specifically modified lymphocytes, preferably T cells. Especially CD8 + T cell lymphocytes are interesting targets in this regard. It has been reported that immune responses derived from single naive, single primary T cells, and single secondary central memory T cells have reached similar phenotypic size and diversity, have undergone comparable stochastic variation, and can ultimately reconstitute immunocompetence against a lethal infection diverse with a bacterial pathogen as measured by target mapping of CD8 + T cells in vivo and their descendants through three generations of single serial cell adoptive transfer and infection-triggered re-expansion (Graef et al., Immunity, 41, 116- 126, 2014). After the development of thymic T cells again from fully mature hematopoietic cells, antigen-specific T cells can be maintained for prolonged periods in an individual, where the antigen may be a foreign antigen, for example, an expressed antigen about a virus or a cancer cell. The targeted modification of similar effector T cells, or their precursors, therefore represents an important strategy for providing suitable T cells for immunotherapy. Naive T cells differentiate through a stage called stem cell memory T cells, which give rise to central memory T cells and effector memory T cells and finally T cells
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146/202 effector cells, in which effector T cells represent terminally differentiated cells which can ultimately recognize and destroy a target cell. Effector memory T cells and effector T cells are subgroups of T cells that have the ability to travel to peripheral tissues. Another subgroup of memory T cells, residing in tissue, are presently suggested, which no longer circulate (cf., for example, Farber et al., Nature Reviews Immunology, 14, 24-35, 2014).
[00218] In addition, cancer immunotherapy has provided some of the first spectacular clinical cases showing that the adoptive transfer of T cells expressing recombinant tumor-reactive receptors can differently cure treatment-resistant malignancies (Brentjens et al., 2013; Grupp et al., 2013; Porter et al., 2011) and that the use of manipulated T cells in adoptive transfer therapies has shown significant promise in the treatment of cancers, particularly hematological cancers. Increasingly, genetically modified T cells of defined phenotypic composition and subgroup are used to increase the success of cancer immunotherapy (see Riddell et al., Cancer J., 20 (2), 141-144, 2014). The use of T cells modified with chimeric antigen receptors as a therapy for hematological malignancies and also for solid tumors is becoming more widespread. To this end, T cells are modified to express tumor-targeted chimeric antigen receptors (CARs) (see, for example, Anurathapan et al., Molecular Therapy, 22, 623-633, 2014). Second-generation CARs are also becoming increasingly important, for example, CD19-targeted CARs that incorporate CD2B or 4-1 BB signaling domains for redirecting and reprogramming T cells to increase their anti-tumor efficacy (see, for example,
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147/202 example, Sjoukje et al., Nature Reviews Drug Discovery, 14, 499-509,
2015).
Therefore, the hybrid RNA / DNA nucleic acid sequences according to the present invention represent an important tool for modifying one or more mammalian cells in vivo or ex vivo, preferably for the treatment of a disease. For example, a lymph cell [I quote, most preferably a T cell or natural killer (NK) from any stage of development to alter a gene expressed by T cells or NK cells to influence proliferation, survival and / or function of T cells or NK cells with high precision in order to avoid off-target effects, which can be detrimental to a therapeutic application of the modified cell or cell population.
[00220] In certain embodiments, the artificial molecular complex according to the present invention is therefore suitable for use in a method of treating a disease, wherein the disease is characterized by at least one genomic mutation and the artificial molecular complex is configured to target and repair at least one genomic mutation. Therefore, a method of treating a disease using the artificial molecular complex according to any of the preceding claims is provided, wherein the disease is characterized by at least one genomic mutation and the artificial molecular complex is configured to target and repair the hair. least one genomic mutation. The therapeutic method of treatment may comprise genetic or genome editing, or gene therapy.
[00221] Suitable cells, particularly for therapeutic approaches, or for modifying a viral genome, include eukaryotic (e.g., animal) and prokaryotic cells and / or cell lines. Non-limiting examples of similar cells or
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148/202 cell lines generated from said cells include COS, CHO cells (for example, CHO-S, CHO-K1, CHO-DG44, CHODUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28 -G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. In certain embodiments, the cell line is a CHO, MDCK or HEK293 cell line. Suitable cells also include stem cells such as, for example, non-human embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells.
[00222] In one aspect, the invention provides a method of treating a subject who needs it, comprising inducing genetic editing by transforming / transfecting the subject with the components of the artificial molecular complex as discussed here, in this patent application, or any of the vectors discussed here, in this patent application, and administering a source of inductive energy to the subject. The invention comprises the use of a polynucleotide or similar vector in the manufacture of a medicament, for example, a similar medicament for treating a subject or for a similar method of treating a subject. The invention comprises the polynucleotide as discussed here, in this patent application, or any of the vectors discussed here, in this patent application, for use in a method of treating a subject who needs it, including inducing genetic editing, in which the method additionally comprises administering a source of inductive energy to the subject. On a
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149/202 aspect, in the method, a repair model is also provided, for example, released by a vector comprising said repair model.
[00223] In one embodiment, minimal lentiviral vectors of non-primates based on the equine infectious anemia virus (VIA) are also contemplated, especially for gene therapy using the artificial molecular complexes of the present invention (see, for example, Balagaan, J Gene Med 2006; 8: 275-285). In another modality, RetinoStat®, a vector of lentiviral gene therapy based on the equine infectious anemia virus is also contemplated, which expresses the angiostatic proteins endostatin and angiostatin which is released through a subretinal injection for the treatment of the macular degeneration network form. related to age (see, for example, Binley et al., HUMAN GENE THERAPY 23: 980-991 (September 2012)) and this vector can be modified for the SSN-RTDD-RT system of the present invention. At present, lentiviral vectors have been disclosed as in the treatment for Parkinson's Disease, see, for example, U.S. Patent Application No. 2012/0295960 A1 and U.S. Patent No. 7,303,910 B2. Lentiviral vectors have also been disclosed for the treatment of eye diseases, see, for example, U.S. Patent Application Nos. 2006/0281180, 2009/0007284, 2011/0117189, 2009/0017543,
2007/0054961, and 2010/0317109. Lentiviral vectors have also been revealed for release to the brain, see, for example, U.S. Patent Application Nos. 2011/0293571, 2011/0293571, 2004/0013648, 2007/0025970, 2009/0111106 and U.S. Patent No. 7,259,015.
[00224] In another embodiment, the artificial molecular complex or components thereof can be administered in liposomes, such as a stable nucleic acid-lipid particle (SNALP) (see,
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150/202 for example, Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3 or 5 mg / kg / day of a specific Cas CRISPR targeted at a SNALP are contemplated. Daily treatment can be for about three days and then weekly for about five weeks. In another embodiment, a specific encapsulated SNALP can be administered by intravenous injection in doses of about 1 or 2.5 mg / kg are also contemplated (see, for example, Zimmermann et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may contain the lipids 3-N - [(wmethoxy poly (ethylene glycol) 2000) carbamoyl] -1,2-dimyristyloxy-propylamine (PEGC-DMA), 1,2-dilinoleyloxy-N, N-dimethyl- 3-aminopropane (DLinDMA), 1,2distearoil-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2: 40: 10: 48 molar ratio (see, for example, Zimmermann et al., Nature Letters, Vol. 441, 2006). In another embodiment, stable nucleic acid-lipid particles (SNALPs) have proven effective in releasing molecules for highly vascularized HepG2-derived liver tumors but not in weakly vascularized HCT-116-derived liver tumors (see, for example, Li , Gene Therapy (2012) 19, 775-780). SNALP liposomes can be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidolcholine (DSPC), Cholesterol and siRNA using a 25: 1 lipid / siRNA ratio and a 48/40/10/2 molar ratio Cholesterol / D-Lin-DMA / DSPC / PEG-CDMA. The resulting SNALP liposomes are about 80 to 100 nm in size.
[00225] In yet another embodiment, an SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3N - [(w-methoxy poly (ethylene glycol) 2000) carbamoyl] -1,2 Petition 870190072502, of 7/29/2019, p. 163/367
151/202 di mi restiloxipropilami na, and 1,2-dilinoleyloxy-3-N, cationic Ndimethylaminopropane (see, for example, Geisbert et al., Lancet 2010; 375: 1896905). A dosage of about 2 mg / kg of
Total SSN / RTDD / RT per dose administered, such as an intravenous bolus infusion.
[00226] Similarly, the artificial molecular complexes according to the present invention can represent a useful tool for modifying genetic material in livestock or cells of other animals. For example, the correction of genetic diseases or editing for favorable characteristics such as meat, milk, for example, milk with a reduced lactose content, or egg production in livestock or poultry.
[00227] In one embodiment, therefore, a method for generating a population of immune cells from an animal comprising introducing a construct according to the present invention into at least one immune cell of interest, in vivo or ex live, to treat a disease, preferably an autoimmune disease, for example, Type I diabetes or rheumatoid arthritis, or a proliferative disease, such as cancer, for example, glioma, melanoma, neuroblastoma, colon, lung cancer, breast and prostate cancer, multi-drug resistant cancers as well as cancers involved with the mutated p53 gene.
[00228] The preferred tissues of most plant species forming targets for genome editing are immature embryos, embryogenic callus, intact plant meristems, pollen, pollen tube or eggs, suspension cells, or other types of cells with regenerative potential . For some plants the preferred tissues may be protoplasts or leaves. Any cell that can be treated and then regenerated into an entire plant can be considered a preferred cell or tissue. Protocols for tissue preparation,
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152/202 regeneration, and DNA release are different depending on the species, the type of tissue, the method of release and other factors. A common release method is to bombard cell particles with gold or tungsten particles coated with DNA or protein. Other methods of release are transformation mediated by polyethylene glycol (PEG), electroporation, viral infection, direct injection into cells, and transformation mediated by Agrobacterium. In some plants it can be released into fertilized eggs by cutting through the style right after fertilization and applying a liquid with the editing reagents into the cut pollen tube. For animal cells, preferably mammalian cells, electroporation, that is, a transfection technology based on the momentary creation of small pores in cell membranes by applying an electrical pulse, may represent a suitable approach for the introduction of at least one molecular complex of according to the present invention. Various cell type-specific protocols for successful direct transfection with a multitude of different cell types, including primary mammalian cells, stem cells and cell lines difficult to transfect, are available to the person skilled in the art, which are suitable as tools of release for the at least one molecular complex according to the present invention. It is important to note that the combination of two or more methods or agents suitable for release can provide superior results depending on the cell type whose genome is to be edited and, therefore, is included within the scope of the present invention.
[00229] In one embodiment, supercharged proteins can be used to release the artificial molecular complex, or components thereof, according to the present invention. Supercharged proteins are a class of proteins produced by
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153/202 genetic engineering or naturally occurring with unusually high net positive or negative theoretical charge and can be employed in the release of one or more artificial molecular complexes or one or more components thereof or one or more nucleic acid molecules encoding for them . Both supernegatively and superpositively charged proteins have a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. The association of charge with these proteins, such as plasmid DNA, RNA, or other proteins, can allow the functional release of these macromolecules within mammalian cells both in vitro and in vivo. David Liu's lab reported the creation and characterization of supercharged proteins in 2007 (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110-10112).
[00230] The non-viral release of RNA and plasmid DNA is of particular interest for the transfer of the artificial molecular complex into mammalian cells are valuable for both research and therapeutic applications (Akinc et al., 2010, Nat. Biotech 26, 561-569). Purified +36 GFP protein (or another superpositively charged protein, for example, +48 GFP) is mixed with RNAs in the appropriate serum-free medium and left to form complex before addition to the cells. The inclusion of serum at this stage inhibits the formation of supercharged protein-RNA complexes and reduces the effectiveness of the treatment. The following protocol has been found to be effective for a variety of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116) (However, pre-experiments varying the dose of protein and RNA should be performed to optimize the procedure for cell lines
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154/202 specific): (1) One day before treatment, laminate 1 x 10 ⁵ cells (eg HEK293, number depending on cell type) per well in a 48-well plate. (2) On the day of treatment, dilute purified +36 GFP protein in serum-free medium to a final concentration of 200 nM. Add RNA to a final concentration of 50 nM. Swirl to mix and incubate at room temperature for 10 min. (3) During incubation, aspirate the cell medium and wash once with PBS. (4) After +36 GFP and RNA incubation, add protein-RNA complexes to cells. (5) Incubate the cells with complexes at 37Ό for 4h. (6) After incubation, aspirate the medium and wash three times with 20 U / mL of heparin PBS. Incubate the cells with serum-containing medium for an additional 48 hours or longer, depending on the test for activity. (7) Analyze cells by immunoblot, qPCR, phenotypic assay, or other appropriate method.
[00231] Another preferred release method for the artificial molecular complex is to assemble the RTDD-RT hybrid nucleic acid in vitro and then load this hybrid into an SSN polypeptide produced in vitro and optionally purified before applying it to the target cells of interest. However, other useful release methods can be used to release the SSN polypeptide and optionally an interaction domain, for example, a monomeric streptavidin, a scFv with a specific specificity, or a DNA binding domain, or a nuclease domain additional as mRNA or as a genetic DNA construct, optionally comprising additional regulatory elements, into at least one target cell for transcription and / or expression in vivo, along with the application of the hybrid nucleic acid simultaneously, before or especially after release of the SSN polypeptide. In the case of a non-covalent association
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155/202 of the RTDD with the RT component, these molecules can also be released separately; in the case that RTDD is a gRNA, the gRNA can be released as RNA or as a DNA expression cassette that can be transcribed in vivo. In cases where at least one SSN polypeptide or at least one gRNA is released as an expression cassette, it may be preferable to express them from a viral DNA or RNA replicon or viral vector, particularly when the target cell it is a plant cell.
[00232] In a preferred embodiment, the at least one artificial molecular complex is associated ex vivo, the different components of the complex, that is, at least one SSN optionally comprising at least one interaction domain, the at least one RTDD and the at least one RT repair model nucleic acid is synthesized, either chemically, or recombinantly, ex vivo / in vitro and the different components are then purified, preferably prior to assembly. An additional purification step can be carried out after the assembly of at least one artificial molecular complex according to the present invention. Methods for purifying nucleic acids, including DNA and RNA, or polypeptides, or ribonucleoprotein complexes - are readily available to the person skilled in the art. The provision of a highly pure and stoichiometric molecular complex, which can be optionally analyzed in vitro, allows the provision of an accurate and highly efficient genome editing tool.
[00233] For modalities based on non-nucleic or non-amino acid based molecules such as RTDDs or interaction domains, for example, biotin (vitamin H) or a derivative thereof, fluorescein, or digoxigenin or any other binding partner cognate for an interaction of SSN-RTDD, or RTDDP, 870190072502, of 7/29/2019, p. 168/367
156/202 interaction domain, or SSN interaction-interaction domain, it is preferable that the RT is synthesized ex vivo and the RT is then chemically linked to the respective molecule.
[00234] In an additional embodiment according to the various aspects according to the present invention, a conventional repair model nucleic acid sequence, either in the form of a plasmid or in the form of a nucleic acid oligonucleotide can be used in addition to RTDD / RT to further increase the efficiency of the targeted genome editing event. Generally, the deciding factor whether a plasmid or other double-stranded DNA repair model is applied or whether a single-stranded oligonucleotide is used as a repair model depends on the size of the intended modification to be introduced. The person skilled in the art can easily define an additional conventional repair model which can be used in addition to the hybrid nucleic acid construct according to the present invention. These conventional repair models can be introduced into at least one target cell of interest by a release vector, for example, a geminiviral vector, if the target cell is a plant cell, or by direct transfection or introduction as also detailed here , in this patent application, for the introduction of the RTDD / RT sequence according to the present invention.
[00235] In one aspect, the invention provides kits containing any one or more of the elements disclosed herein, in this patent application. In some embodiments, the kit comprises a vector system as taught here, in this patent application, and instruction for using the kit. The elements can be provided individually or in combinations, and can be provided in any suitable container, such as a vial, bottle, or tube. Kits can include gRNA and a chain
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157/202 protective bond not bound to stabilize the gRNA. Kits can include the gRNA as RTDD interacting directly with an RT of interest and optionally with an additional protective chain attached at least partially to the guide sequence. Therefore kits can include gRNA in the form of a partially double-stranded nucleotide sequence. In some embodiments, the kit includes instructions in one or more languages, for example, in more than one language. The instructions may be specific to the applications and methods described here, in this patent application.
[00236] In a further aspect according to the present invention, therefore, a kit is provided comprising at least one component and preferably all components of at least one artificial molecular complex of the present invention, wherein the at least one molecular complex can be provided as a pre-assembled complex, or preferably where the at least one molecular complex can be provided in the form of its separate constituents, comprising at least one SSN polypeptide, or an expressible sequence encoding the same, at least one RTDD sequence and at least one repair model nucleic acid sequence. The separate provision of the different constituents of the molecular complex, preferably in the form of a dry or lyophilized powder for nucleic acid sequences, guarantees greater stability of the nucleic acid sequences particularly of the RTDD / RT construct, particularly if sequences are provided of RNA being much less stable than polypeptides within the kit. At least one SSN protein and optionally at least one interaction domain interacting with or connected to it can be released within a suitable storage buffer, for example, comprising 300 mM NaCI, 10 mM Tris-HCI, 0, 1
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158/202 mM EDTA, 1 mM OTT, 50% Glycerol, pH 7.4 at 25Ό for a Cas9 polypeptide. The kit may additionally comprise a suitable reaction buffer including suitable ions, for example,
Mg2 + for a Cas9 enzyme, required for the activity of a respective CRISPR polypeptide.
[00237] In some embodiments, a kit comprises one or more reagents for use in a process using one or more of the elements described here, in this patent application. The reagents can be provided in any suitable container. For example, a kit can provide one or more reaction or storage buffers. The reagents can be provided in a form that is usable in a particular test, or in a form that requires the addition of one or more other components before use (for example, in concentrated or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector in order to operationally link the guide sequence. and a regulatory element. In some embodiments, the kit comprises a homologous recombination model polynucleotide. In some embodiments, the kit comprises one or more of the vectors and / or one or more of the polynucleotides described here, in this patent application. The kit can advantageously make it possible to provide all elements of the systems of the invention.
[00238] Alternatively, the kit can comprise the components
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159/202 of SSN as lyophilized mRNA or lyophilized protein, respectively. In an additional embodiment according to this aspect, the kit may comprise an additional component providing a suitable delivery vehicle or delivery system in addition to a component comprising the one or more components of the SSN as a molecular complex. In an additional embodiment according to this aspect, at least one SSN polypeptide and at least one RTDD / RT sequence are presented as at least two components, the more than one SSN and / or RTDD / RT being mutually compatible. The at least one SSN polypeptide can be presented as a vector to be transformed or transfected into a cell of interest, whereas at least one RTDD / RT sequence can be presented as a separate component. A kit according to the present disclosure can therefore be suitable for the simultaneous or subsequent use of the different components in the event that more than one component is present. Optionally, a kit according to this aspect can comprise instructions for use, particularly instructions for specific use for a target cell to be edited. In an additional preferred embodiment in accordance with this aspect of the present invention, the kit is specifically developed to provide a feature development kit for a specific plant of interest including specific tools to obtain the desired feature modification. According to this modality, the kit comprises a specific repair model, which is configured to transfer the characteristic of interest or to treat a disease of interest, or to modify a target DNA sequence of interest into a target locus of DNA of interest in a cell, preferably a mammalian cell or a plant cell. In addition, the kit comprises a
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160/202 suitable SSN enzyme, or two SSN nickases, associated as complex with at least one RTDD, wherein the RTDD comprises at least a first portion of the sequence interacting directly with at least one SSN, a second portion of the sequence configured to interact directly with at least one repair model nucleic acid (RT) sequence, and wherein the at least one RTDD is configured to be associated with or be able to associate with a repair model carrying the specific characteristic of interest.
[00239] A kit according to a modality is both a plant cell as well as a specific characteristic, and the use of the referred kit makes it possible to quickly target and modify a genomic DNA locus of interest in order to obtain the development of the characteristic. In one embodiment, RTDD is a gRNA, in which the components of the gRNA are already designed to interact with PAM motifs and a CRISPR enzyme of interest and the repair model provided presents the sequence to be inserted or modified in a convenient way.
[00240] In one aspect according to the present invention there is therefore provided a plant, plant cell, plant material, or derivative, or progeny thereof comprising or edited by at least one artificial molecular complex according to present invention. In a further aspect according to the present invention there is provided a plant, a plant cell, a plant material, or a derivative, or a progeny thereof that has been modified with at least one artificial molecular complex.
[00241] In a still further aspect according to the present invention there is provided a method of modifying at least one target DNA sequence, comprising the following steps: (i)
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161/202 providing at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising at least one genomic complementarity sequence and at least one target DNA sequence in a genomic region of interest; (ii) providing at least one artificial molecular complex as defined herein, in this patent application; (iii) contacting at least one artificial molecular complex with at least one target DNA sequence under conditions suitable to effect (a) interaction of at least one site-specific nuclease with at least one target DNA sequence; and (b) complementary base pairing of at least one repair model nucleic acid sequence with at least one genomic complementarity sequence to perform recognition of at least one complementarity sequence and induction of at least one DNA break by at least at least one site-specific nuclease, wherein the at least one repair model nucleic acid sequence directs homologous repair at the site of at least one target DNA sequence; and (iv) obtaining at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising a modification in at least one target DNA sequence.
[00242] Due to the fact that the artificial molecular complex can be used within any cell type of interest, it is possible to design an SSN / RTDD / RT pair for the modification of any genomic region, including episomics or epigenetics of an organism of interest , comprising prokaryotic, eukaryotic or viral DNA target sequences or epigenetic sequences of interest. For modalities of modifying the genome of a virus, it is appropriate to transfer the viral genome, or the relevant part of it, into a vector of interest and to propagate and modify the viral genome within a suitable host cell (for example, a cell
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162/202 prokaryotic or a eukaryotic cell) carrying the vector comprising the viral genome, or the relevant part of it.
[00243] A prokaryotic cell as used here, in this patent application, refers to a single-celled organism that lacks a membrane-bound nucleus (karyon), mitochondria, or any other membrane-bound organelle and comprises archaea and bacteria.
[00244] A viral genome can be derived from any virus comprising a genome encoded by RNA or DNA.
[00245] In one embodiment, at least one repair model nucleic acid sequence and / or at least one repair model docking domain of the artificial molecular complex is / are provided for at least one prokaryotic or eukaryotic cell independently of at least one site-specific nuclease of at least one molecular complex and the at least one artificial molecular complex is assembled, or partially assembled, within at least one cell and / or prokaryotic / eukaryotic / o genome, or viral.
[00246] In one embodiment, at least one RTDD / RT sequence of the artificial molecular complex is provided for at least one prokaryotic or eukaryotic cell independently of at least one SSN polypeptide of at least one molecular complex and at least an artificial molecular complex is assembled, or partially assembled, within at least one prokaryotic or eukaryotic cell.
[00247] The at least one artificial molecular complex, as detailed above, can be proposed as a complex assembled in vitro which is then introduced into at least one target cell of interest. Alternatively, part or all of at least one SSN polypeptide and / or at least one RTDD sequence and / or
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163/202 to at least one repair model nucleic acid sequence can be inserted as a RNA or genetic DNA construct and can be produced in vivo so that the final assembly of at least one molecular complex occurs in vivo. In a preferred embodiment, the at least one molecular complex is associated ex vivo and the at least one molecular complex comprising at least one SSN polypeptide, at least one guide nucleic acid sequence and at least one repair model nucleic acid sequence it is then simultaneously provided to the at least one cell by a suitable release vector enabling the functional introduction of at least one molecular complex into at least one target cell comprising at least one target DNA sequence of interest.
[00248] In another preferred embodiment, at least one SSN and optionally at least one interaction domain are provided as a fusion protein on a plasmid to be produced within a cell comprising a target DNA sequence to be modified. The additional components of the artificial molecular complex can then be produced ex vivo. For example, an inducible vector system can be used to produce at least one SSN and optionally at least one interaction domain. As soon as a sufficient level of expression is obtained, the RTDD / RT complex can be introduced into a target cell and the artificial molecular complex according to the present invention can be assembled in situ.
[00249] In another embodiment, the at least one complete artificial molecular complex is an artificial molecular complex assembled ex vivo.
[00250] Adequate conditions or reaction conditions as referred to here, in this patent application, in
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164/202 context of the methods according to the present disclosure, refer to conditions which allow both the growth and development of a cell or an organism, including prokaryotic or eukaryotic cells, being transformed or manufactured and the conditions necessary to obtain or stable integration or transient introduction of a genetic construct of interest in at least one cell or organism of interest. The conditions for promoting prokaryotic or bacterial growth and / or transformation are known to the person skilled in the art (see also: Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Laboratory Press). Conditions for promoting the growth of animal cells and / or for introducing genetic material into animal cells, particularly mammalian cells, are available to the person skilled in the art for a variety of different cell lines (see Green and Sambrook supra). The conditions for promoting the growth and development of plants or plant cells, including but not limited to temperature, light, water, oxygen, mineral nutrients and soil support, which may vary for different plant species and can be readily determined by the expert in the field. with knowledge of the disclosure provided here, in this patent application. The additional suitable conditions for obtaining stable integration or transient introduction of at least one molecular complex of interest depend on the transformation method selected for the introduction of at least one molecular complex of interest, the stage of development of the plant material or plant cell to be transformed and at least one molecular complex of interest to be introduced. The aforementioned suitable conditions can be defined by the person skilled in the art in light of the present disclosure by defining the appropriate conditions for the methods in combination with
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165/202 exemplary molecular complexes and release vectors and suitable release techniques as disclosed and claimed herein, in this patent application.
[00251] In an embodiment according to the method above of the present invention, the at least one eukaryotic cell is a plant cell, preferably a plant cell of a plant selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor , Saccharum officinarium, Zea spp., Including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum tausus, A Daucus glochidiatus, Beta spp., Including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Cactus, Solanum tuber, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalay Himalayas, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicariap. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Goscine, Glycine, Gosscine, Glycine. , Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the plants mentioned above.
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166/202 [00252] With respect to plant cells as targets, for example, a variety of transformation and / or transfection methods are available to the person skilled in the art in the field. For corn protoplasts, for example, a suitable method is disclosed in Sheen, J. 2002 (A transient expression assay using maize mesophyll protoplasts). For Arabidopsis protoplasts, a suitable protocol is available at: doi.org/10.1038/ngrot.2007.199 or can be retrieved from http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199. html.
For tobacco and other dicotyledon protoplasts, a suitable protocol is available at www.plantphysiol.org/cgi/doi/10.1104/pp.112.205179. Therefore, the person skilled in the art, having knowledge of the present disclosure and being aware of the cited protocols, can define a suitable method for the introduction of a molecular complex according to the present invention into a plant protoplasty derived from a monocot or a dicot plant .
[00253] Protoplasts are very useful for testing genetic editing technologies and reagents, but for the regeneration of genetically edited plants they are not always the preferred cell type, since very few plant species regenerate effectively from protoplasts. In these cases the preferred tissues for most plant species are immature embryos, embryogenic callus, fertilized embryos, intact plant meristems, pollen, pollen tube or eggs, embryogenic suspension cells, or other cell types with regenerative potential. A common physical release method is to bombard cell particles with gold or tungsten particles coated with DNA or protein, whereas a common biologically assisted method uses Agrobacterium or a
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167/202 viral vector (modified) as disclosed here, in this patent application.
[00254] Meristematic cell (s) as referred according to the present disclosure belong to a type of tissue within a plant which is also referred to as meristem or formative or exchange tissue. Like stem cells in animal organisms, meristematic plant cells representing undifferentiated cells have intrinsic capacity to develop and differentiate into specialized cell types, depending on genetic predisposition and additional environmental and developmental factors. In plant organisms, meristems are present not only during the development of embryos, but can be found throughout the life cycle of a plant so that a targeted genetic modification of meristematic cells or tissues according to the present disclosure is not restricted to plant embryos or seedlings, but can also be carried out preferably on larger seedlings and more mature plants, for example, when targeting meristems which build the base for the plant's reproductive organs, for example, tassel or ear in corn.
[00255] According to a modality according to the various aspects according to the present disclosure, a meristematic cell can be a mature or immature plant cell of a plant embryo or a plant seedling comprising at least one meristematic cell or a meristematic tissue.
[00256] For certain genome editing approaches, stable integration of the molecular complex encoding one or more expression cassettes may be desirable, where a transgenic organism carrying a desired construct of interest, or part of it, can inherit an inserted construct in a way
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168/202 stable for the progeny of a plant cell of interest initially transformed or transfected. The referred stable integration can occur within any genomic region of an organism, preferably a eukaryotic organism, including the nuclear genome as well as the extra nuclear genome, including the plastid genome.
[00257] A transient introduction may be desirable, if a certain effect is desired by the introduction of a molecular complex of interest, or part of it, but the construct per se should not be transmitted to a progeny of the cell initially. Due to regulatory reasons, a similar approach may be especially suitable for certain applications, particularly with cells, tissues, organs or plant materials as a structure comprising the target DNA sequence to be modified.
[00258] The term targeted integration or functional integration as used here, in this patent application, refers to the integration of a genetic construct of interest within at least one cell, which allows for transcription and / or translation and / or activity catalytic and / or binding activity, including the binding of one nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within at least one cell. Where relevant, functional integration occurs in a given cell compartment of at least one cell, including the nucleus, cytosol, mitochondria, chloroplast, vacuole, membrane, celual wall and the like. Consequently, the term functional integration - in contrast to the term stable integration detailed above - implies that the molecular complex of interest is introduced into at least one cell by any means of transformation, transfection or transduction by
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169/202 biological means, including transformation by Agrobacterium, or physical means, including particle bombardment, as well as the subsequent step, in which the molecular complex exerts its effect within or on at least one cell into which it has been introduced. Depending on the nature of the genetic construct to be introduced, the effect referred to naturally can vary and including, alone or in combination, among others, the transcription of a DNA encoded by the genetic construct to a ribonucleic acid, the translation of an RNA into a sequence of amino acids, the activity of an RNA molecule within a cell, comprising the activity of a guide RNA, or a miRNA or a siRNA for use in RNA interference, and / or a binding activity, including the binding of a molecule of nucleic acid to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within at least one cell, or including the integration of a sequence released through a vector or genetic construct, either transiently or in a stable mode. The said effect may also comprise the catalytic activity of an amino acid sequence representing an enzyme or a catalytically active portion thereof within at least one cell and the like. The said effect obtained after functional integration of the molecular complex according to the present disclosure may depend on the presence of regulatory sequences or localization sequences which are comprised by the genetic construct of interest as is well known to the person skilled in the art.
[00259] As detailed above, the methods according to the present invention targeting pluripotent or multipotent cells provide the advantage that both transformation and further development of at least one transformed cell, particularly a meristematic cell, can occur
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170/202 in planta eliminating the need for laborious stages of in vitro cultivation for the regeneration of a plant or plant material. However, in certain modalities, it may be convenient to explant or dissect a cell, tissue, organ or plant material for cultivation, screening or further experimentation depending on specific needs. Various methods for in vitro cultivation of a cell, tissue, organ or plant material are available to the person skilled in the art.
[00260] Therefore, a stable integration may be desirable, where a transgenic plant carrying a desired construct of interest, or a part of it, is inserted in a stable manner and the inserted construct or part of it is transmitted to the progeny of a cell of interest initially transformed. The referred stable integration can occur within any genomic region of the plant, including the nuclear genome as well as the extranuclear genome, including the plastid genome of a plant cell. In addition, the artificial molecular complexes according to the present invention can be used to create an epigenetic modification. In another aspect, the present invention provides a method of functional evaluation and screening of genes. Therefore, the artificial molecular complex of the present invention can be used to precisely release functional domains, to activate or repress genes or to change the epigenetic state by precisely altering the methylation site on a specific locus of interest. A method of the invention can be used to create a plant, animal or cell that can be used to model and / or study genetic or epigenetic conditions of interest, such as through a model of mutations of interest or a model of disease. As used herein, in this patent application, disease refers to a disease, disorder, or indication in a subject. For example, a
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The method of the invention can be used to create an animal or cell that comprises a modification in one or more nucleic acid sequences associated with a disease, or a plant, animal or cell in which the expression of one or more acid sequences nucleic diseases associated with a disease are altered. A similar nucleic acid sequence can encode a disease-associated protein sequence or it can be a disease-associated control sequence. Therefore, it should be understood that in the embodiments of the invention, a plant, a subject, a patient, an organism or a cell can be a non-human subject, patient, organism or cell. Therefore, the invention provides a plant, animal or cell, produced by the present methods, or a progeny thereof. The progeny can be a clone of the plant or animal produced, or it can result from sexual reproduction by crossing with other individuals of the same species to introgress additional desirable characteristics within their progeny. The cell can be provided in vivo or ex vivo in the case of multicellular organisms, particularly animals or plants. In the case where the cell is in culture, a cell line can be established if appropriate culture conditions are met and preferably if the cell is suitably adapted for this purpose (for example a stem cell). Bacterial cell lines produced by the invention are also envisaged. Consequently, cell lines are also expected.
[00261] A transient introduction may be desirable, if a certain effect is desired, for example, a silencing effect, targeted manipulation, comprising a knock-in or a knock-out, by introducing a genetic construct of interest , or a part of it, but the construct per se does not
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172/202 must be transmitted to a progeny of the initially transformed cell.
[00262] In yet another embodiment of the above aspect, according to the present invention, the introduction of at least one molecular complex of interest, or parts thereof including gRNA and / or RT, is conducted using a medium selected from the group consisting of a device suitable for particle bombardment, including a gene gun, including a portable gene gun (eg Helios® Gene Gun System, BIO-RAD) or a stationary gene gun, transformation, including transformation using Agrobacterium spp. or using a viral vector, microinjection, electroporation, whisker technology, including silicon carbide whisker technology, and chemical, for example, using calcium phosphate, dendrimers, liposomes or cationic polymers, and non-chemical, for example using electroporation, sonoporation, optical transfection using a laser, fusion of protoplasts, impalefection, hydrodynamic genetic release of DNA by injecting a release construct into an organ, preferably within the liver, of an animal, preferably a rodent animal , transfection, or a combination thereof.
[00263] In certain embodiments, the at least one eukaryotic cell is a meristematic plant cell, and the plant cell, after the introduction of the artificial molecular complex according to the present invention, is further grown under suitable conditions until the development stage is reached maturity of the inflorescence, in order to obtain a plant or a vetetal material comprising a modification of interest mediated by at least one molecular complex according to the present invention. Several protocols are available, for example,
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173/202 to the person skilled in the art for the production of germinable and viable pollen from in vitro cultivated corn tassels, for example, in Pareddy DR et al. (1992) Maturation of maize pollen in vitro. Plant Cell Rep 11 (10): 535-539. doi: 10.1007 / BF00236273, Stapleton AE et al. (1992) Immature maize spikelets develop and produce pollen in culture. Plant Cell Rep 11 (5-6): 248-252 or Pareddy DR et al. (1989) Production of normal, germinable and viable pollen from in vitro-cultured maize tassels. Theor Appl Genet 77 (4): 521-526. These protocols are, among others, based on the excision of the tassel, superficial sterilization and culture in a medium with kinetin to promote the growth and maturation of the tassels. After spikelets are formed, a continuous anther harvest can be carried out. After extrusion, the anthers will be desiccated until the pollen leaves. Alternatively, the anthers can be desiccated and the pollen is poured into a liquid medium that is subsequently used to pollinate the ears. [00264] Inflorescence maturity as used here, in this patent application, refers to the state when the immature inflorescence of a plant comprising at least one meristematic cell has reached a stage of development, when a mature inflorescence is obtained, that is, an inflorescence staminate (male) or pistil inflorescence (female), and therefore a gamete of pollen (male) or egg (female) or both is present. The referred stage of the reproductive phase of a plant is especially important, since the plant material obtained can be used directly for the pollination of an additional plant or for fertilization with pollen from another plant.
[00265] In an additional embodiment according to the method above of the present invention, the modification of at least one target DNA sequence is a genome editing approach selected from the group consisting of increased production,
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174/202 tolerance to abiotic stress, including water stress (due to drought), osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, saline or flooding stress, tolerance to biotic stress including insect tolerance, tolerance bacteria, virus tolerance, fungus tolerance or nematode tolerance, herbicide resistance, including glyphosate, glufosinate, acetolactate synthase (ALS) inhibitors, and Dicamba, lodging resistance, flowering time, crack resistance, seed color, endosperm composition, nutritional content, modification of phenotypic markers, or metabolic engineering, including genome editing to allow a molecular pharming approach in at least one plant cell. Phenotypic markers can be preferred targets for co-editing approaches, for example, to monitor the efficiency of editing.
[00266] In another embodiment, characteristic development is carried out for a prokaryotic cell or viral genome, for example, to provide a prokaryotic cell with a metabolic pathway confidently modified to produce a product of interest, or to provide an attenuated viral genome. .
[00267] In another embodiment according to the method above of the present invention, modification of at least one target DNA sequence is a genome editing approach for ex vivo modification of an immune cell in at least one eukaryotic cell, so preferably a mammalian cell, preferably a mammalian leukocyte, to obtain a modified cell suitable for treating a viral disease or for immunotherapy, especially immunotherapy for cancer.
[00268] In a preferred embodiment, the above method according to the present invention is a method for modifying
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175/202 a eukaryotic cell, preferably at least one plant cell, in a targeted manner to provide a genetically modified plant, preferably non-transgenic, in which the method, among others, may be a method for characteristic development. For example, a highly site-specific substitution of 1, 2, 3 or more nucleotides can be introduced in the sequence encoding a plant gene in order to produce substitutions of one or more amino acids that will confer tolerance to at least one herbicide such as glyphosate , glufosinate, Dicamba or an acetolactate synthase (ALS) inhibiting herbicide. In addition, in another embodiment, substitutions of one or more amino acids in the coding sequence of a nucleotide-binding site rich leucine repeating gene (NBS-LRR) that will alter the protein's pathogen recognition spectrum to optimize the resistance to plant disease. In yet an additional embodiment, a small enhancer sequence or transcription factor binding site can be modified into an endogenous promoter of a plant gene or can be introduced into the promoter of a plant gene in order to alter the profile of a plant gene. expression or strength of the plant gene regulated by the promoter. The expression profile can be changed through various modifications, introductions or deletions in other regions, such as intrans, 3 'untranslated regions, cis- or trans- reinforcer sequences. In yet an additional embodiment, the genome of a plant cell, preferably a meristematic plant cell, can be modified in such a way that the plant resulting from the modified meristematic cell can produce a chemical substance or a compound of agronomic or pharmaceutical interest, for example, insulin or insulin analogs, antibodies, a protein with an enzyme function of interest, or any other suitable pharmaceutically relevant compound
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176/202 as a medicine, as a dietary supplement, or as a health care product.
[00269] In an additional aspect, editing a feature according to the methods of the present invention provides a method of editing a feature to perform the treatment of a disease and / or condition and / or prevent infection / infestation by insects in a plant comprising modification of chromosomal or extrachromosomal genetic material from said plant by using any of the preceding methods. Non-limiting examples of diseases and / or conditions treatable by the invented methods include Anthracnose Stalk Rot, Aspergillus Ear Rot, Common Corn Ear Rots, Corn Ear Rots, Common Rust of Corn, Diplodia Ear Rot, Diplodia Leaf Streak, Diplodia Stalk Rot, Downy Mildew , Eyespot, Fusarium Ear Rot, Fusarium Stalk Rot, Gibberella Ear Rot, Gibberella Stalk Rot, Goss's Wilt and Leaf Blight, Gray Leaf Spot, Head Smut, Northern Corn Leaf Blight, Physoderma Brown Spot, Pythium, Southern Leaf Blight, Southern Rust, and Stewart's Bacterial Wilt and Blight, and combinations thereof.
[00270] Non-limiting examples of insects that cause, directly or indirectly, diseases and / or conditions treatable by the invented methods include Armyworm, Asiatic Garden Beetle, Black Cutworm, Brown Marmorated Stink Bug, Brown Stink Bug, Common Stalk Borer, Corn Billbugs, Corn Earworm, Corn Leaf Aphid, Corn Rootworm, Corn Rootworm Silk Feeding, European Corn Borer, Fall Armyworm, Grape Colaspis, Hop Vine Borer, Japanese Beetle, Scouting for Fall Armyworm, Seedcorn Beetle, Seedcorn Maggot, Southern Corn Leaf Beetle, Southwestern Corn Borer, Spider Mite, Sugarcane Beetle, Western Bean Cutworm, White Grub, and Wireworms, and combinations thereof. The methods of the invention are also suitable for preventing infections and / or infestations of a plant by
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177/202 any of these insects.
[00271] Non-limiting examples of characteristics that can be introduced by this method are resistance or tolerance to pests by insects, such as root-eating larvae, stem borers, moth caterpillars (cutworms), beetles, aphids, leafhoppers, weevils, mites and bedbugs. These can be produced by modifying plant genes, for example, to increase a plant's inherent resistance to insect pests or to reduce its attractiveness to said pests. Other characteristics may be resistance or tolerance to nematodes, bacterial, fungal or viral pathogens or their vectors. Still other characteristics can be more efficient use of nutrients, such as use of improved nitrogen, improvements or introductions of efficiency in nitrogen fixation, increase in photosynthetic efficiency, such as conversion of plants C3 to C4. Still other characteristics can be increased tolerance to abiotic stressors such as temperature, water supply, salinity, pH, tolerance to extremes in sunlight exposure, efficiency of nitrogen use, efficiency of phosphorus use, efficiency water use and crop or biomass production. Additional characteristics may be characteristics related to the taste, appearance, nutrient or vitamin profiles of edible or foodable portions of the plant, or they may be related to the longevity of storage or the quality of these portions. Finally, traits can be related to agronomic qualities such as lodging resistance, shattering, flowering time, ripening, emergence, harvest, plant structure, vigor, size, production, and other characteristics. In order to obtain the above characteristic modification, the method according to the present invention comprises material modification
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178/202 genetic chromosomal or extrachromosomal of a plant or plant cell using any of the preceding methods. [00272] In an embodiment according to the above method according to the present invention, the target cell is a prokaryotic cell and the modification comprises at least one modification of a genomic target region of interest to at least one prokaryotic cell, wherein the modification is suitable for modulating or increasing the resistance of the bacteria against biotic or abiotic stress, including resistance against antibiotics, or where the modification is suitable for increasing the phage resistance of at least one prokaryotic cell. In another embodiment, the modification comprises inserting a gene of interest into the DNA target site of at least one prokaryotic cell of interest, for example, to insert a sequence encoding a fluorescent marker protein or other selectable marker within at least one target DNA site of interest. In another embodiment, the modification comprises knocking-out, that is, the deletion of at least one target DNA site of interest in at least one prokaryotic cell. As prokaryotic cells will no longer differentiate, but they can directly inherit at least one modification introduction of interest to their progeny and since prokaryotic cells generally have a very short generation time compared to eukaryotic cells, a modification can be made as introduced by at least one RTDD / RT in the form of at least one artificial molecular complex according to the present invention quickly, and the resulting population of modified cells can be obtained and analyzed in a very short period of time.
[00273] In certain embodiments, the above method according to the present invention may additionally comprise the
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179/202 following step: (v) identification and / or selection of at least one prokaryotic or eukaryotic cell comprising the modification in at least one target DNA sequence, or identification of a modification to a viral genome as propagated in a prokaryotic cell or eukaryotic.
[00274] Methods for analyzing or identifying a modification according to the present disclosure as carried out in the genome of at least one prokaryotic or eukaryotic cell or a viral genome are known to the person skilled in the art and comprise, but are not limited to, chain reaction. polymerase (PCR), including but not limited to real-time quantitative PCR, multiplex PCR, RT-PCR, nested PCR, analytical PCR and the like, microscopy, including bright and dark field microscopy, scatter staining, phase contrast, fluorescence, confocal , differential interference contrast, deconvolution, electron microscopy, UV microscopy, IR microscopy, scanning probe microscopy, analysis of a cell's metabolite, analysis of an altered resistance spectrum of a modified cell, RNA analysis, analysis of proteome, functional assays to determine a functional integration, for example, of a marker gene or of a transgene of interest, or a knock-out, analysis by Southern-Blot, sequencing, including deep sequencing and combinations thereof. Cells comprising the desired modification can then be selected for further culture or any other downstream manufacturing step.
[00275] In a further aspect according to the present invention there is provided a method for making a plant or plant cell comprising the following steps: (i) carrying out a method of modifying at least one target DNA sequence in a cell eukaryotic as detailed above, where the
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180/202 at least one eukaryotic cell is a plant cell; (ii) obtaining at least one plant or one progeny from it from at least one plant cell from step (i); (iii) optionally: determining the modification in at least one target DNA sequence in at least one cell of at least one plant or a progeny thereof.
[00276] Cells, tissues, organs and plant materials suitable for carrying out this aspect are detailed above. The term manufacturing according to the present disclosure should be considered in a general way and includes any form of genetic manipulation performed on the genetic material of a plant or plant cell. The provision of at least one artificial molecular complex comprising at least one RTDD / RT sequence comprising at least one repair model docking domain and at least one repair model nucleic acid and at least one SSN polypeptide, optionally comprising one domain of interaction, can occur in a mode to allow transient action or stable integration, or a combination thereof, of the different components as detailed above. Preferably, the at least one artificial molecular complex, or the different components thereof, are provided in a transient manner so that no integration of any of those effector components occurs, such as, including a sequence encoding an acid RNA. guide nucleic, a sequence encoding a repair model nucleic acid DNA, and a sequence encoding a CRISPR polypeptide, within the genome of the target cell of interest.
[00277] In an embodiment according to the above manufacturing method according to the present invention, at least one plant or plant cell is selected from among a plant
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181/202 monocotyledonous or a dicotyledonous, preferably, where the plant is selected from the group consisting of Zea spp., Including Zea mays, Nicotiana benthamiana, or Beta spp, including
Beta vulgaris, or Secale ssp., Including Secale cereal, or Triticum ssp., Including Triticum aestivum.
[00278] As detailed from the beginning to the end of the present disclosure, the methods according to the present invention are suitable and can be adapted for target cells belonging to all realms of life, such as the use of an RTDD / RT construct, which is associated in a functional way in combination with an appropriate site-specific nuclease interacting with the RTDD is independent of species and cell, as long as there is a homologous recombination mechanism for DNA repair in the cell, even dictated by the covalent or non-covalent form of at least one gRNA and at least one RT. What needs to be determined individually for each target cell and for each target are (i) the site-specific nuclease or catalytically active fragment thereof and whether the use of an interaction domain, for example, as a fusion protein, may be appropriate; (ii) a suitable RTDD-SSN or RTDD-interaction domain pair allowing direct interaction of the referred components by recognition of cognate link partners; and (iii) a suitable RT and its connection to the RTDD, where the RT design is relevant to introduce a customized repair in a target DNA sequence of interest cleaved by at least one SSN of the artificial molecular complex and optionally (for nucleases CRISPR) (iv) a gRNA and the CRISPR polypeptide, which must be compatible as detailed above; (v) a correspondence of the gRNA of interest with a PAM site within the target region of DNA of interest; and (vi) the target DNA sequence and the target modification to be introduced. For any genome
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182/202 publicly available sequences, the design of suitable nucleic acid sequences can therefore be produced in silico based on the disclosure of the present invention.
[00279] In a further aspect according to the present invention, the use of at least one RTDD / RT sequence according to the present invention is provided, or the use of an artificial molecular complex according to the present invention for editing genome in a prokaryotic cell or a eukaryotic cell. In one embodiment of this aspect, the use is for a eukaryotic cell, preferably a fungal cell or organism, an animal or plant cell or organism, or a viral organism as propagated in a prokaryotic cell or a eukaryotic cell.
[00280] According to the various aspects and modalities according to the present invention, a eukaryotic cell or a method or use for modifying some eukaryotic cells, including stem cells, does not explicitly include any human cloning process, a process for modifying the genetic identity of the germline of human beings or the use of human embryos, or a method requiring the destruction of human embryos to obtain cells from the same cells. Specifically, human germline cells or human embryos are therefore specifically excluded as target cells or organisms to be modified with artificial molecular complexes or by the methods according to the present invention.
[00281] The present invention is further described with reference to the following non-limiting examples.
Examples [00282] The present invention is further illustrated by
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183/202 non-limiting examples that follow.
[00283] Example 1: Hybrid nucleic acid sequence as a pair
RTDD / RT suitable to be combined with a Cas polypeptide or
Cpf 1 or Argonauta [00284] In an experiment, the caudate sgRNA or sgDNA are hybridized through both complementary base pairing and RNA-DNA or DNA-DNA binding with a single chain repair model. For covalent association, synthesized DNA oligonucleotides are covalently linked to the 3 'end of the RNA / DNA oligonucleotides using the ssRNA ligase manufacturer's protocol. For non-covalent association, RNA / DNA and DNA with partially complementary sequences are mixed and allowed to form a complex through Watson-Crick base pairing. The success of hybridization can be determined in gel change assays. The treatment of aliquots of the hybrid nucleic acid with RNase and DNase enzymes before the gel change assays indicates that part of the hybrid nucleic acid is composed of RNA and part of the DNA for the experiments using sgRNAs. The hybrid nucleic acid is then complexed with recombinant Cas9 protein or another CRISPR-derived or Argonaut-derived nuclease. The success of complex formation can be verified by treating with proteinase K, RNase, DNase and a pseudo treatment, and observing the relative gel exchange patterns. Recombinant Cas polypeptides were produced and subsequently purified either by an external commercial entity or by internal capacity. Different hybrid nucleic acid sequence architectures between a guide nucleic acid sequence such as RTDD and a tested repair model (RT) nucleic acid sequence are shown in Figures 1 and 2.
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184/202 [00285] Example 2: In vitro cut of a DNA target by a Cas9 protein complex with an RNADNA hybrid nucleic acid [00286] In one experiment, the functionality of the Cas protein as a site-specific endonuclease was tested when used with the described hybrid nucleic acid technology. A linearized plasmid containing at least one target site for sgRNA was mixed with a Cas9-sgRNA-RT complex as described in the present invention. After incubation under conditions suitable for nuclease activity, including the correct pH, temperature and cofactors and the like, which are well known to the person skilled in the art for various CRISPR nucleases and variants thereof, the target DNA plasmid was passed over a agarose gel and observed for band sizes indicating cut at the expected target site. In vitro cleavage of the target DNA indicated that the RT associated with the sgRNA as a charge did not interfere with the normal function of the Cas9 complex as a site-specific endonuclease.
[00287] Example 3: In vivo editing by Cas9 protein forming complex with a hybrid RNA-DNA nucleic acid [00288] In order to demonstrate that a target gene can be edited in vivo by a linerate complex comprising Cas9 protein and a nucleic acid hybrid RNA-DNA, a non-functional tdTomato gene contained within a transformed plasmid was repaired by exchanging a single nucleotide to restore the fluorescent signal of the tdTomato gene. In order to determine the optimal use for editing by providing an ssDNA repair model with complementarity to the target chain or to the non-target chain, complexes were compared loading repair models from either chain.
[00289] The hybrid RNA / DNA polypeptide Cas nucleic acid complex obtained in Example 1 was used to repair a target
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185/202 of episomic plasmid, encoding a tdTomato gene with a single point mutation from A to T that creates an early stop signal at the position of codon 51. This plasmid was introduced into a corn protoplasty system along with the editing complex comprising Cas9 protein and an RNADNA hybrid nucleic acid via PEG-mediated release or electroporation. A single chain repair model is then linked to sgRNA through complementary base pairing. The repair model is complementary to the region of 80 base pairs downstream and -40 base pairs upstream of the cutting site. The success of the next edition results in some cells showing a fluorescence phenotype of tdTomato due to the repair of the tdTomato gene in at least one plasmid contained within them. The relative efficiency of editing with different repair models can therefore be easily assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00290] Example 4: In vivo editing by Cas9 protein forming complex with an RT attached to the RNA component by covalent bonding or associated by complementary base pairing [00291] In order to demonstrate editing with hybrid nucleic acid molecules manufactured in various ways , the optimal conditions identified in Example 3 were used to evaluate the repair of the same episomic plasmid target with covalent binding of hybrid nucleic acids or pairing of non-covalent bases of the repair model to the sgRNA.
[00292] If a marker is used, particularly a fluorescent marker, successful editing will result in some cells showing a fluorescence phenotype due to the repair of the fluorescence encoding gene, such as a tdTomato gene, in at least one plasmid contained within them. The relative
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186/202 efficiency of editing with the different repair models can then be assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00293] Example 5: In vivo editing by Cas9 protein complexing with a hybrid nucleic acid formed by binding the RT to the 5 'or 3' end of the sgRNA [00294] In one example, the method described in Example 3 can be used to identify a preference for the repair model hybridized or linked to the 5 'or 3' end of the sgRNA. The covalence of the preferred bond determined in Example 4 can be used here. Based on the results presented in Tsai et al. (Dimeric CRISPR RNA-guided Fokl nucleases for highly specific genome edition ”, Nature Biotechnology, 32, 569-576 (2014), doi: 10.1038 / nbt.2908) and in addition, Shechner et al. (Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display ”, Nature Methods, 12 (7), 664-670 (2015), doi: 10.1038 / nmeth.3433), a 3 'fusion is expected to be preferred .
[00295] The success of the edition results in some cells showing a fluorescence phenotype, such as a tdTomato phenotype, due to the repair of the tdTomato gene in at least one plasmid contained within them. The relative efficiency of editing with the different repair models can then be assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00296] Example 6: Determination of optimal ligand length between sgRNA and repair model for in vivo editing by Cas9 protein forming complex with a hybrid nucleic acid [00297] In one example, an increasing ligand length in increments of 50 pairs of bases up to a length of 500 base pairs between sgRNA and repair model was used to identify
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187/202 optimum conditions for homologous recombination to repair the target described in Example 3. Employing a set of ligand lengths will help determine the necessary flexibility needed within the hybrid to overcome the protein's target chain geometry. This is particularly necessary when working with different CRISPR nucleases and, therefore, specific gRNAs and individual repair models (RTs) to coordinate the interaction of the molecular complex and to ensure that the CRISPR complex, also in the presence of RT, can exert its effect. . The conditions of Example 3 were used together with the optimized parameters determined within Examples 3 to 5. The linker was DNA with complementarity for sequence close to the target gene.
[00298] The success of the edition will result in some cells showing a fluorescence phenotype of tdTomato, in case a tdTomato marker is used, due to the repair of the tdTomato gene in at least one plasmid contained within them. The relative efficiency of editing with the different ligand lengths can then be assessed by measuring the abundance of fluorescent cells resulting from each treatment. Likewise, any other selectable marker of interest can be used including any fluorescent marker suitable for a cell type of interest, antibiotic markers, marker sequences, regulatory sequences and the like.
[00299] Example 7: Determination of the optimal repair model configuration for in vivo editing by Cas9 protein forming complex with a hybrid nucleic acid [00300] In order to demonstrate editing with single-stranded and double-stranded repair models, the test in vivo described in Example 3 was used for a relative comparison of the two configurations. Single-chain repair models are expected to be better
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188/202 based on lower molecular weight and higher published editing rates with short ssDNA oligos than with short dsDNA oligos. However, the use of a double-stranded repair model may be necessary in cases where large strings need to be edited or inserted. The optimal conditions of Examples 4 and 6 can be used in this example.
[00301] The success of an editing event results in some cells showing a fluorescence phenotype, such as a tdTomato phenotype, due to the repair of the tdTomato gene in at least one plasmid contained within them. The relative efficiency of editing with the different repair models can then be assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00302] Example 8: In vivo editing of a chromosomal target by Cas9 protein forming a complex with a hybrid RNA-DNA nucleic acid [00303] In one example, the method optimized by Examples 3 to 7 can be used to make edits to a chromosomal target gene. Here, a transgenic corn plant with a stable insertion of the tdTomato early stop codon cassette was used to demonstrate the utility of the invention for a chromosomal target. The editing success resulted in some cells showing a tdTomato fluorescence phenotype due to the repair of the tdTomato gene integrated in the genomic DNA. Editing efficiency was assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00304] Example 9: In vivo insertion of a genetic cassette into a chromosomal target by Cas9 protein forming a complex with a hybrid nucleic acid of RNA-DNA [00305] In order to demonstrate the utility of the invention for
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189/202 insertion of a full-length gene into a chromosomal target, a tdTomato fluorescent reporter gene and terminator were integrated into the corn hmg13 gene, resulting in a fluorescent tdTomato signal due to the expression triggered by the endogenous promoter for hmg13. The results can demonstrate that long insertions can be made using the method of the invention and that they will help to optimize the conditions for said insertion. [00306] The successful editing results in some cells showing a tdTomato fluorescence phenotype due to the insertion of the tdTomato gene within the hmg13 target and the subsequent expression of tdTomato protein can be confirmed. The efficiency of the corresponding edition for each cell type tested can then be assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00307] Example 10: Use of a cell-penetrating peptide to release Cas9 protein into plant cells forming a complex with a hybrid RNA-DNA nucleic acid [00308] The optimal system identified in Examples 8 or 9 was used in this example to to test the effectiveness of PEG-based transformation versus transformation with a cell-penetrating peptide (CPP). Previous publications and requirements suggest that the use of CPPs for release will allow the introduction into cells with a cell wall of the Cas9 protein forming a complex with a hybrid nucleic acid of RNA-DNA. Thus, CPPs were used within a Cas fusion protein or linked to Cas via a disulfide bond formed between an N-terminal cysteine on the Cas protein and an N-terminal cysteine on the CPP. Free CPPs can also be used to assist the import of the Cas complex and nucleic acid through transient binding over the nucleic acid chain. Starting CPPs can
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190/202 include the HIV TAT peptide (see, for example, SEQ ID NOs: 17 and 18), or a sequence derived therefrom and / or a sequence of (Arg) g. Effectiveness can be tested using the optimized methods of
Examples 3 to 9 through tdTomato expression successfully in a protoplast system.
[00309] Example 11: Additional CRISPR Nucleases [00310] As detailed above, the hybrid nucleic acid sequences according to the present invention are suitable for a variety of CRISPR nucleases from different CRISPR systems. For any effector nuclease, for example, Cas9 or Cpf 1, the optimal conditions and lengths of the gRNA and RT will have to be evaluated as detailed in Examples 1 to 10 above in order to obtain optimal results for a genome editing event from interest for each cell type of interest. In addition, first experiments with Cas9 nickases were conducted in the same way, as detailed above using more than one gRNA and or one or two individual RTs associated with at least one of the gRNAs. Early results demonstrate that this appears to be a promising approach to the accuracy of genome editing in eukaryotic cells as well.
[00311] Example 12: Animal cell constructs [00312] The method of the invention can be used in eukaryotic cells as long as they are capable of homologous recombination. In a first example, murine T cells or T cell precursors were modified in vitro to modulate them to be suitable for cancer immunotherapy. It can be demonstrated that the hybrid nucleic acid constructs according to the present invention, when specifically optimized (codon optimization) and designed (PAMs, target sites) for an animal system can be used for high precision editing of
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191/202 genome in a type of eukaryotic animal cell of interest. The modification of an expressed gene regulating T cell proliferation or function using the method described in this invention, therefore, can be used for therapy, particularly in a mammal, and more particularly to treat a disease or disorder in a subject by modifying a cell type of interest with the constructs according to the present invention.
[00313] Example 13: Transformation / transfection of exposed immature tassel tissue [00314] As detailed above, a variety of physical / mechanical as well as biological means have been described for the transformation of plant cells, tissues, organs or whole plants or parts thereof for introducing genetic material into a plant or target plant structure. These methods are likewise suitable for introducing at least one hybrid RNA / DNA nucleic acid sequence and / or at least one gRNA, and / or at least one repair model, and / or at least one CRISPR polypeptide according to with the present invention. After you have exposed and therefore obtained a meristematic cell, for example, a tassel tissue from a male corn plant, the following methods can be applied to transform this tissue:
[00315] With reference to biological media, plant tissues or cells thereof can be transformed with Agrobacterium, including transformation mediated by Agrobacterium tumefaciens or by Agrobacterium rhizogenes. This type of transformation is generally known to a person skilled in the art (see, for example, Jones, HD et al., Review of methodologies and a protocol for the Agrobacterium-med iated transformation of wheat ', plant methods, 2005; or Frame, BR et al., Agrobacterium tumefaciensmed iated transformation of maize embryos using a standard binary
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192/202 vector system, Plant, 2002). For this purpose, an Agrobacterium culture comprising a construct of interest is, for example, grown overnight at 28Ό in fluid Luria Broth medium containing a suitable antibiotic, 10 mM MES and 200 mM ACE. The next day, the culture overnight is centrifuged at 4,400 rpm for 15 min and the supernatant is discarded. Then the pellet is again centrifuged for 15 min at 4,400 rpm for 2 min and the remaining supernatant is discarded. The pellet is resuspended (5 ml H ₂ O, 10 mM MES, 10 mM MgCl ₂ + 20 μΜ ACE). The optical density at 600 nm is adjusted to 1.5. The possibly diluted suspension can then be used later.
[00316] Another possibility for the transformation of meristematic cells or tissues of a plant through biological means is the use of viral vectors. Viral vectors have the advantage that they can be introduced as either DNA or RNA and to a target structure of the plant of interest. In addition, viral vectors or plant viruses have the ability to spread within different cells and tissues.
[00317] For the purposes of the present invention, virus particles, in vitro virus transcripts or Agrobacteria carrying a virus encoding T-DNA can be introduced into the target structure of a plant of interest through filtration (vacuum and not vacuum) ). Alternative experiments can be performed using plant sap. For this purpose, either tobacco or spinach can be infected with the virus of interest to subsequently isolate said virus of interest from the plant's sap to infect another target plant structure, especially meristematic cells or tissues from different plants with the sap from the plant containing the virus.
[00318] Despite biological means of transforming tassel structures of interest, physical / mechanical means can be used
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193/202 for transformation in addition to particle bombardment.
[00319] A suitable method is microinjection. Microinjection can be used for any type of tested meristem structure, preferably using a microscope with a micromanipulator. Due to the size of a certain meristematic structure such as tassel or ear, microinjection of meristems can be conducted under microscopic control or, in the case where the target structures are large enough, without microscopic assistance. The injection can be conducted using a variety of methods for a variety of different target molecules to be introduced into the target structure of a target plant of interest including double-stranded plasma DNA, linear double-stranded DNA, RNA and proteins as well as particles virus in liquid solution. These different molecules can be applied with the help of some micro- or nano-needles which assist in the injection of the target molecules into the cell or meristem structure of interest. First, the target molecules are coated on the needle, which is then inside the cell or meristem structure of interest.
[00320] Another suitable means is bombardment of particles, for example, using a particle release system, this method being further disclosed above.
[00321] A further development of this technology is the use of a combination of silicon carbide (SiC) whiskers (for example, Silar® Silicon Carbide Whisker) and microinjection. For this purpose, double-stranded DNA (optionally plasmid), linear double-stranded DNA, RNA, protein, or a molecular ribonucleo-complex according to the present invention, or virus particles are precipitated onto the silicon carbide whisker a be injected through a microinjection needle into the structure or cell
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194/202 meristem of interest. This technique has the advantage that it is not only possible to transfect a single cell, but there is the possibility of penetrating different cells in parallel due to the spread of whiskers. In addition, the cells are less destroyed, since the needle does not need to penetrate into the cell and the whiskers are quite small in size.
[00322] Example 14: Means for detecting a modification [00323] Any transient or stable modification as introduced within at least one target DNA sequence according to the present invention can be detected using a fluorescence detection means, in the case a fluorescent reporter is used. As the tissues of the tassels such as anthers and dry pollen have a strong autofluorescence, other means will be used for these cells and tissues. Detection can therefore be performed and confirmed by additional molecular methods, such as PCR, including enrichment PCR, digestion by PCR, a combination of enrichment PCR with digestion by PCR, quantitative PCR, or sequencing, or RT-PCR, including deep or next generation sequencing or Southern or Northern blot analysis. Protein levels can be analyzed by Western-Blotting and the like. In the event that a phenotypically detectable characteristic has been introduced into at least one cell of interest, it was also possible to perform a test to detect whether the referred characteristic, for example, a resistance, a fluorescence, a morphological mutant phenotype, or any additional characteristic , is present or absent in at least one modified cell or in a progeny or derivative thereof. The above detection methods are known to the person skilled in the art.
[00324] As usual configuration for the analysis of a stable integration event in different target plants and cells of the same
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195/202 can be conducted as follows: First, DNA and / or RNA are extracted from different materials, including, for example, tassel, anther or pollen tissue / cells transformed with different constructs encoding a fluorescent protein, for example , a red fluorescent protein. In short, samples can be analyzed by quantitative PCR (qPCR). From the samples above, several samples will show a light fluorescent (red) signal, that is, a very intense fluorescent signal, which is indicative of a positive event and which can then be selected. From these samples, cDNA will be generated including controls without reverse transcriptase to exclude that the latest results are not associated with undigested DNA. Of the samples with a positive DNA signal used for transcription measurement, several samples may have a clear transcription and others a potential transcription (within the limits of what can be clearly measured).
[00325] Example 15: Cas9 and scFv fusion protein [00326] In one experiment, a Cas9 nuclease fusion protein like SSN and a single chain antibody against fluorescein as the interaction domain can be expressed in vitro or in vivo and exposed to a FAM-labeled oligonucleotide to act as a repair model. RT was synthesized and covalently linked to FAM as a repair model docking domain. Editing efficiency was measured by a fluorescent signal indicating repair or sequence-based measurements of the repair frequency as detailed above. The SSN-interaction pair of a Cas9 and a scFv with a specific affinity for a selected ligand, for example, FAM, can therefore be produced and purified separately and can then be cross-linked or connected, or the SSN and the interaction domain (AI) can be produced as a fusion molecule. Depending on the test, the SSN-IA molecule can be
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196/202 transfected into a cell or added to a test as a protein, or the construct can be introduced into a target cell over a vector (inducible or active in a constitutive way) to be transcribed and translated in vivo. In addition, the sequence encoding SSN-IA can be introduced into a target cell comprising a target DNA sequence of interest to be modified as an RNA construct to be translated in vivo. Examples of SSN-IA fusion molecules according to the present invention combining the functionality of a CRISPR-derived SSN with the extremely high binding affinity of a specialized protein for its cognate partner are shown with SEQ ID NO: 44 (construct of fusion Cas / mSA) and SEQ ID NO: 45 (Cas / scFv (FAM) fusion construct). Figure 4 A to C schematically illustrates a genome engineering approach using fusions of an SSN and a monomeric streptavidin or an scFv like IA. Noteworthy, the use of monomeric streptavidin or scFvs or any other AI or RTDD is not restricted to the use of an Argonauta or CRISPR nuclease.
[00327] Example 16: Nucleic acid binding by a Cas9 fusion protein bound to scFv [00328] In order to demonstrate the ability of the Example 15 fusion protein to bind a single-stranded or double-stranded repair model, the test described ligation will be repeated with a fluorescein-labeled oligonucleotide (FAM). Fluorescein-labeled oligonucleotides can be obtained commercially. The success of an interaction can be tested by co-migration of protein, DNA, and the fluorescent dye and the corresponding increase in molecular weight. The functionality of the nuclease part of the fusion protein will be tested using an in vitro divination test of a specific guide RNA and a linearized plasmid harboring the
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197/202 corresponding target. After incubation under conditions suitable for nuclease activity, including the correct pH, temperature and cofactors and the like, which are known to the person skilled in the art for various CRISPR nucleases and variants thereof, the target DNA plasmid was passed over a gel agarose and observed for band sizes indicating cut at the expected target site. In vitro cleavage of the target DNA indicated that the RT associated with the nuclease did not interfere with the normal function of the Cas9 complex as a site-specific endonuclease.
[00329] Example 17: Cas9 and mSA2 fusion protein [00330] In one experiment, a Cas9 nuclease fusion protein and a modified streptavidin marker (based on SEQ ID NO: 34) was expressed and exposed to a labeled oligonucleotide with biotin acting as a repair model, biotin acting as RTDD and the oligonucleotide representing a RT. Editing efficiency was measured by a fluorescent signal indicating repair or measurements based on the repair frequency sequence.
[00331] Example 18: Nucleic acid binding by a Cas9 fusion protein linked to mSA2 [00332] In order to demonstrate the ability of the Example 17 fusion protein to bind a single-stranded or double-stranded repair model, the test described ligation was repeated with a biotin-labeled oligonucleotide. Biotin-labeled hiigonucleotides can be obtained commercially or generated using terminal deoxynucleotidyl transferase. The success of an interaction can be tested by protein and DNA co-migration and the corresponding increase in molecular weight. The functionality of the nuclease part of the fusion protein will be tested using an in vitro cleavage test of a specific guide RNA and a linearized plasmid harboring the corresponding target. After incubation under
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198/202 suitable for nuclease activity, including the correct pH, temperature and cofactors and the like which are known to the person skilled in the art for various CRISPR nucleases and variants thereof, the target DNA plasmid was passed over an agarose gel and observed for band sizes indicating cut at the expected target site. In vitro cleavage of the target DNA indicated that the RT associated with the nuclease did not interfere with the normal function of the Cas9 complex as a site-specific endonuclease.
[00333] Example 19: In vivo editing of an episomic target by Cas9 fusion protein forming a complex with a FAM-labeled repair model nucleic acid or biotin to restore genetic functionality [00334] In order to demonstrate that a target gene can be edited in vivo by a released complex comprising the Cas9 protein and a FAM-labeled or biotin nucleic acid, a non-functional tdTomato gene contained within a transformed plasmid was repaired by exchanging a single nucleotide to restore the gene's fluorescent signal tdTomato. In order to determine the optimal use for editing by providing an ssDNA repair model with complementarity to the target chain or the non-target chain, complexes carrying repair models from either chain are compared.
[00335] The fusion protein complexing with the nucleic acid of Example 16 or 18 respectively was used to repair an episomic plasmid target, encoding a tdTomato gene with a single point A to T mutation that creates an early stop signal at codon of position 51. This plasmid was introduced into a corn protoplast system together with the editing complex comprising the Cas9-ScFV or Cas9mSA2 fusion protein and a FAM-labeled or biotin nucleic acid via
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199/202 PEG-mediated release. The editing success then results in some cells showing a fluorescence phenotype of tdTomato due to the repair of the tdTomato gene in at least one plasmid contained within them. The relative efficiency of editing with the different repair models can therefore be easily assessed by measuring the abundance of fluorescent cells resulting from each treatment.
[00336] Example 20: In vivo editing of a chromosomal target by Cas9 fusion protein forming a complex with a FAM-labeled repair model nucleic acid or with biotin to integrate DNA sequence into a specific locus [00337] In order to demonstrate that a target gene can be edited in vivo by a released complex comprising Cas9 protein and a nucleic acid labeled with FAM or with biotin, a specific known DNA sequence, will be integrated into a specific site within the genomic DNA.
[00338] The Cas9 fusion protein and a single chain variable fragment with affinity for fluorescein (Example 16) or the Cas9 fusion protein and the modified streptavidin (Example 18) was expressed and exposed to labeled repair model DNA and used to integrate a known DNA sequence into a genomic locus. The success of the edition will be analyzed by repairing the fluorescent signal indication or molecular tests at the target site.
[00339] Example 21: Nucleic acid binding by an Argonaut fusion protein bound to scFv [00340] In order to demonstrate the ability to bind a repair model nucleic acid to a non-CRISPR nuclease, a binding test was performed showing weight gain in a co-migration study using a FAM-labeled repair oligo nucleic acid and a nuclease fusion protein
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200/202
Argonaute (see SEQ ID NO: 46) and a single chain variable fragment with affinity for FAM (see SEQ ID NOs: 43 and 45). Likewise, an Argonaut SSN can be linked to a monomeric streptavidin (see SEQ ID NOs: 42 and 44) as a binding complex for an RT. The functionality of the nuclease portion of the fusion protein was tested using an in vitro divination test of a specific guide nucleic acid and a linearized plasmid harboring the corresponding target. After incubation under conditions suitable for nuclease activity, including the correct pH, temperature and cofactors and the like, which are known to the person skilled in the art for various non-CRISPR nucleases and variants thereof, the target DNA plasmid was passed over a agarose gel and observed for band sizes indicating cut at the expected target site. In vitro cleavage of the target DNA indicated that the RT associated with the nuclease did not interfere with the normal function of the Argonauta complex as a site-specific endonuclease.
[00341] Example 22: In vivo editing of a chromosomal target by an Argonaut fusion protein forming a complex with a FAM-labeled repair model nucleic acid to integrate DNA sequence into a specific locus [00342] In order to demonstrate that a target gene can be edited in vivo by a released complex comprising the non-CRISPR protein Argonauta nuclease and a nucleic acid labeled with FAM or biotin, a known and specific DNA sequence will be integrated into a specific site within the genomic DNA. [00343] The Argonauta nuclease fusion protein and a single chain variable fragment with affinity for fluorescein (see Example 21) was expressed and exposed to a labeled repair model DNA and used to integrate a known DNA sequence into a genomic locus. The success of the edition will be analyzed by repair
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201/202 of fluorescent signal indication or molecular tests at the target site.
[00344] Example 23: CRISPR nuclease fusion protein (as
Cas9 or Cpf1) and an RTDD1 [00345] In order to demonstrate that the tethering strategy is working, a CRISPR nuclease purified as Cas9 or Cpf1 was fused with an RTDD1, in this case it is tied to a single chain variable fragment (SEQ ID NO: 54) and expressed in E. coli bacteria. It was operated on a denaturing SDS gel, with a continuous gradient (4 to 10%) and shows the quantity and purity of the protein. The protein was stained in this gel. The right panel of Figure 5 shows the tethering. This is a 4% non-denaturing acrylamide gel (Blue Native PAGE) and here the DNA is stained using Red Gel. The FAM-labeled repair model (RTDD2-) was either incubated in the nuclease buffer without or with the nuclease- RTDD1 shown in the left panel of Figure 5. If the protein was present, tying occurred as seen by DNA being detected at a higher molecular weight level (arrow in Figure 5).
[00346] Example 24: Detection of HDR events [00347] In order to demonstrate that sequencing of the next generation, more specifically deep amplicon sequencing, is capable of detecting the HDR event at the target site, the encoded nuclease (in this case it was a CRISPR nuclease) fused to the variant streptavidin was transformed on a plasmid together with the repair model. The repair model had a 5 'biotin marker and was released as a single chain oligonucleotide. Twenty-four hours after transformation, protoplasts were collected and DNA was extracted. The target site was amplified using a set of primers that were designed not to overlap with the homology arms of the repair model. Line 4 in Figure 6 shows the correct HDR event. The event replaces the string
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AAGGTGCTCGGCCCCGAGCTC (SEQ ID NO: 52; encoding the KVLGPEL amino acid sequence) with AAGTGGTCCAGCGCCGCGACCTAGCTC (SEQ ID NO: 53; encoding the KWSSAAT-L amino acid sequence). SEQ ID NO: 51 is the complete repair model demonstrating that the homology arms are not extending beyond the amplicon, meaning that PCR artifacts with the remaining repair model are unlikely.
[00348] Example 25: Tethering of the repair model improves the efficiency of the HDR [00349] For this experiment, the components of Example 24 were transformed into corn leaf protoplasts. In the case of mooring, the nuclease (in this case it was a CRISPR nuclease) was fused to a native streptavidin sequence. In any case, the nuclease was released as a plasmid. The repair model DNA was released as an oligonucleotide with a 5 'biotin marker. Twenty-four hours after transformation, protoplasts were collected and DNA was extracted. The target site was amplified using a set of primers that were designed to not overlap with the homology arms of the repair model. Deep amplicon sequencing (see Example 24) and subsequent computational analysis allows quantification of INDEL and HDR events at the target site. The HDR frequency was normalized to the INDEL frequency as a measure of the occurrence of double strand breaks. The average HDR frequency increased from 0.92% (± 0.06%) without mooring to 1.26% (± 0.06%) when the repair model is tied to the nuclease (Figure 7).

权利要求:
Claims (23)
[1]
1. Artificial molecular complex, characterized by the fact that it comprises:
(a) at least one site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding it, and interacting directly with it (b) at least one repair model docking domain (RTDD), or a nucleic acid sequence encoding it, where the repair model docking domain is configured to interact directly with at least one repair model (RT) nucleic acid sequence;
(c) optionally comprising at least one interaction domain (IA), or a nucleic acid sequence encoding the same, wherein the at least one interaction domain is interacting directly with the at least one site-specific nuclease or the catalytically active fragment of the same, and in which at least one interaction domain is configured to provide at least one of the features selected from the group consisting of (i) interaction with at least one repair model docking domain; and / or (ii) interaction with at least one repair model nucleic acid sequence; and / or (iii) sequence-specific interaction with genomic DNA;
wherein the at least one repair model nucleic acid sequence comprises at least a portion being complementary to at least one genomic complementarity sequence, and where at least one repair model nucleic acid sequence is configured to mediate repair of a
Petition 870190072502, of 7/29/2019, p. 216/367
[2]
2/11 target DNA sequence.
2. Artificial molecular complex according to claim 1, characterized in that the site-specific nuclease, or the nucleic acid sequence encoding it, is selected from at least one among a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN , a ZFN, a meganuclease, a restriction endonuclease, including Fokl or a variant thereof, or two site-specific cut endonucleases, or a variant or a catalytically active fragment thereof.
[3]
3. Artificial molecular complex according to claim 1 or 2, characterized by the fact that at least one repair model docking domain, or the nucleic acid sequence encoding the same, is selected from at least one among biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with at least one acid sequence nucleic repair model, a streptavidin, or a variant thereof, preferably a monomeric streptavidin, an avidin, or a variant thereof, an affinity marker, preferably a streptavidin marker, an antibody, a variable fragment single-stranded (scFv), a single-domain antibody (nanobody), anticalin, an Agrobacterium VirD2 protein or a domain thereof, a VPg of Picornavirus, a topoisomerase or a domain thereof, a PhiX174 phage A protein, a PhiX A * protein, a VirE2 protein or a domain of the same, or digoxigenin.
[4]
4. Artificial molecular complex according to any of the preceding claims, characterized by the fact that the
Petition 870190072502, of 7/29/2019, p. 217/367
3/11 at least one interaction domain, or the nucleic acid sequence encoding it, is selected from at least one of a DNA binding domain, a streptavidin, or a variant thereof, preferably a monomeric streptavidin, avidin, or a variant thereof, an affinity marker, a biotinylation signal, a biotin acceptor site, a streptavidin marker, an antibody, a single chain variable fragment (scFv), a single domain antibody (nanobody) , an anticalin, biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetrazolium (XTT), a guide nucleic acid sequence specifically configured to interact with the at least one repair model nucleic acid sequence, an AgrDobacterium VirD2 protein or a domain thereof, a Picornavirus VPg, a topoisomerase or a mezzanine domain sma, a PhiX174 phage protein, a PhiX A * protein, a VirE2 protein or a domain thereof, or digoxigenin.
[5]
Artificial molecular complex according to any one of the preceding claims, characterized in that the at least one site-specific nuclease and / or at least one repair model nucleic acid sequence and / or the at least one domain of interaction comprises at least one nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence or a chloroplast localization sequence.
[6]
Artificial molecular complex according to any one of the preceding claims, characterized in that the at least one repair model nucleic acid sequence comprises at least one end portion, preferably the 3 'end, wherein this end is end portion not
Petition 870190072502, of 7/29/2019, p. 218/367
4/11 interacts with any other component of the artificial molecular complex and is therefore configured to hybridize to at least one genomic complementarity sequence in order to mediate repair of the target DNA sequence, and / or in which at least one sequence of repair model nucleic acid is provided as a plasmid.
[7]
7. Artificial molecular complex according to any one of the preceding claims, characterized by the fact that at least one site-specific nuclease or the catalytically active fragment thereof, or the sequence encoding it, is selected from a CRISPR nuclease, of preferably between a Cas nuclease or a Cpf1, or a Fokl nuclease, or a catalytically active fragment thereof, and the at least one interaction domain, or the sequence encoding the same, is selected from a single chain variable fragment or a monomeric streptavidin.
[8]
8. Artificial molecular complex according to any one of the preceding claims, characterized in that the complex comprises at least one guide nucleic acid sequence representing at least one repair model docking domain, each of which at least a guide nucleic acid sequence comprises (i) a first portion of the sequence that is complementary to a target recognition DNA sequence, and (ii) a second portion of the sequence, wherein the second portion of the sequence is configured to interact with the at least one site-specific nuclease, and (iii) in which at least one guide nucleic acid sequence is physically associated with at least one repair model nucleic acid sequence and therefore forms a sequence
Petition 870190072502, of 7/29/2019, p. 219/367
5/11 hybrid nucleic acid comprising or consisting of at least one RNA or DNA and at least one additional DNA nucleic acid sequence, and (iv) optionally comprising a linker region between at least one guide nucleic acid sequence and to at least one repair template nucleic acid sequence, preferably wherein the repair template nucleic acid sequence is associated with the guide nucleic acid sequence at the 3 'end of the guide nucleic acid sequence, and / or wherein the repair template nucleic acid sequence is associated with the 5 'end of the guide nucleic acid sequence, and / or where the repair template nucleic acid sequence is located within the guide nucleic acid sequence.
[9]
Artificial molecular complex according to any one of the preceding claims, characterized in that the at least one repair model nucleic acid sequence and / or at least one guide nucleic acid sequence comprises a nucleotide sequence selected from among a nucleotide sequence that occurs naturally or not naturally, including a synthetic nucleotide sequence, optionally comprising backbone and / or base modifications, wherein the guide nucleic acid sequence comprises a single-stranded RNA or DNA nucleotide sequence or partially single-stranded, and wherein the at least one repair template nucleic acid sequence comprises a single-stranded or a double-stranded DNA nucleotide sequence.
[10]
10. Artificial molecular complex according to any of the preceding claims, characterized by the fact that at least one site-specific nuclease, or the sequence encoding the same, and the at least one interaction domain, or the sequence
Petition 870190072502, of 7/29/2019, p. 220/367
6/11 encoding the same, and / or at least one repair model docking domain, or the sequence encoding the same, are connected by at least one linking domain.
[11]
11. Artificial molecular complex according to any one of the preceding claims, characterized by the fact that at least one site-specific nuclease or the catalytically active fragment thereof, or the sequence encoding it, is independently selected from the group consisting of a Cas polypeptide from Streptococcus spp., including Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria spp., including Neisseria meningitides, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Flavio , Sphaerochaeta, Azospirillum, Gluconacetobacter, Roseburia, Parvibaculum, Nitratifractor, Mycoplasma and Campylobacter, Candidatus Micrarchaeum acidiphilum ARMAN-1, Parcubacteria (GenBank: APG80656.1), Sulfolobus spp., Including Sulfolobus.17: or REY15A (GenBank: ADX84852.1), a Cpf1 polypeptide from an archaea or a ba matter, including a Cpf1 polypeptide from Acidaminococcus spp., including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., Including Lachnospiraceae bacterium ND2006, Lachnospiraceae bacterium MC2017, Lachnospiraceae bacterium MA2020, Butyrivibrio proteoclasticus, Candidatus spp.
GW2011_GWC2_44_17, Smithella sp. SCADC, Smithella sp. SC_K08D17, Francisella spp., Including Francisella novicida U112, Eubacterium eligens, Prevotella spp., Or Porphyromonas spp., Or an Argonauta nuclease of Natronobacterium gregoryi (GenBank:
Petition 870190072502, of 7/29/2019, p. 221/367
7/11
AFZ73749.1), Microcystis aeruginosa (Reference Sequence NCBI:
WP_012265209.1 or NCBI Reference String:
WP_002747795.1 or NCBI Reference String:
WP_012265209.1), Halogeometricum pallidum (GenBank: ELZ29017.1), Natrialaba asiatica (Reference Sequence NCBI: WP_006111085.1), Natronorubrum tibetense (Reference Sequence NCBI: WP_006090832.1), Natrinema pellirubrum18 .1), or Synechococcus spp. (NCBI Reference Sequence: WP_011378069.1) or variants and / or functional fragments and / or combinations thereof, including nickases, or nucleases lacking endonucleolytic activity.
[12]
12. Artificial molecular complex according to any one of the preceding claims, for use in a method of treating a disease, characterized by the fact that the disease is distinguished by at least one genomic mutation and the artificial molecular complex is configured to target and repair at least one genomic mutation.
[13]
13. Method of treating a disease using the artificial molecular complex as defined in any of the preceding claims, characterized by the fact that the disease is distinguished by at least one genomic mutation and the artificial molecular complex is configured to target and repair the hair least one genomic mutation.
[14]
14. Plant, plant cell, plant material, or derivative, or progeny thereof characterized by the fact that it comprises or is edited by at least one artificial molecular complex as defined in any of claims 1 to 11.
[15]
15. Method of modifying at least one target DNA sequence, characterized by the fact that it comprises the following
Petition 870190072502, of 7/29/2019, p. 222/367
8/11 steps:
(i) providing at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising at least one genomic complementarity sequence and at least one target DNA sequence in a genomic region of interest;
(ii) providing at least one artificial molecular complex as defined in any one of claims 1 to 11;
(iii) contacting at least one artificial molecular complex with at least one target DNA sequence under conditions suitable to effect (a) interaction of at least one site-specific nuclease with at least one target DNA sequence; and (b) complementary base pairing of at least one repair model nucleic acid sequence with at least one genomic complementarity sequence to perform recognition of at least one complementarity sequence and induction of at least one DNA break by at least at least one site-specific nuclease, wherein the at least one repair model nucleic acid sequence directs homologous repair at the site of at least one target DNA sequence; and (iv) obtaining at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising a modification in at least one target DNA sequence.
[16]
16. Method according to claim 15, characterized in that the at least one repair model nucleic acid sequence and / or the at least one repair model docking domain of the artificial molecular complex is / are provided for to at least one prokaryotic or eukaryotic cell independently of at least one site-specific nuclease of at least one molecular complex and at least
Petition 870190072502, of 7/29/2019, p. 223/367
9/11 an artificial molecular complex is assembled, or partially assembled, within at least one prokaryotic, eukaryotic, or viral cell and / or genome.
[17]
17. Method according to claim 15, characterized in that the at least one artificial molecular complex is an artificial molecular complex assembled ex vivo.
[18]
18. Method according to any one of claims 15 to 17, characterized in that the at least one eukaryotic cell is a plant cell, preferably a plant cell of a plant selected from the group consisting of Hordeum vulgare, Hordeum bulbusom , Sorghum bicolor, Saccharum officinarium, Zea spp., Including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium marach, Horach Aegilops tauschii, Daucus glochidiatus, Beta spp., Including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanumumumum, Cananumumumum, Lymphaticumera, Solanumumumum, Lymphaea Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis th aliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica vuca, nica. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine, Glycine.
Petition 870190072502, of 7/29/2019, p. 224/367
11/10
Japanese lotus, Torenia fournieri, Allium cepa, Allium fistulosum,
Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the plants mentioned above.
[19]
19. Method according to claim 18, characterized by the fact that the modification of at least one target DNA sequence causes the edition of a characteristic selected from the group consisting of increased production, tolerance to abiotic stress, including water stress ( drought), osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, saline stress or flooding, tolerance to biotic stress including insect tolerance, bacterial tolerance, virus tolerance, fungal tolerance or tolerance nematode, herbicide resistance, including glyphosate, glufosinate, acetolactate synthase (ALS) inhibitors, and Dicamba, lodging resistance, flowering time, crack resistance, seed color, endosperm composition, nutritional content, or metabolic engineering, including genome editing to allow a molecular pharming approach in at least us a plant cell.
[20]
20. Method according to any one of claims 15 to 19, characterized in that it further comprises the following stage:
(v) identifying and / or selecting from at least one prokaryotic, eukaryotic, or viral cell and / or genome comprising the modification in at least one target DNA sequence.
[21]
21. Method for manufacturing a plant or plant cell characterized by the fact that it comprises the following steps:
(i) perform a method as defined in any of the
Petition 870190072502, of 7/29/2019, p. 225/367
11/11 claims 15 to 20, wherein the at least one eukaryotic cell is a plant cell;
(ii) obtaining at least one plant or a progeny thereof from at least one plant cell in step (i);
(iii) optionally: determining the modification in at least one target DNA sequence in at least one cell of at least one plant or a progeny thereof.
[22]
22. Method according to claim 21, characterized by the fact that at least one plant or plant cell is selected from a monocot or a dicot plant, preferably, in which the plant is selected from the group consisting of Zea spp ., including Zea mays, Nicotiana benthamiana, or Beta spp, including Beta vulgaris, or Secale ssp., including Secale cereal, or Triticum ssp., including Triticum aestivum.
[23]
23. Use of at least one artificial molecular complex as defined in any one of claims 1 to 11, characterized in that it is for genome engineering in a cell and / or genome and / or prokaryotic, eukaryotic, or viral organism, so preferred in a plant cell or organism.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

AR006928A1|1996-05-01|1999-09-29|Pioneer Hi Bred Int|AN ISOLATED DNA MOLECULA CODING A GREEN FLUORESCENT PROTEIN AS A TRACEABLE MARKER FOR TRANSFORMATION OF PLANTS, A METHOD FOR THE PRODUCTION OF TRANSGENIC PLANTS, A VECTOR OF EXPRESSION, A TRANSGENIC PLANT AND CELLS OF SUCH PLANTS.|

---

US7022896B1|1997-04-04|2006-04-04|Board Of Regents Of University Of Nebraska|Methods and materials for making and using transgenic dicamba-degrading organisms|

---

GB9720465D0|1997-09-25|1997-11-26|Oxford Biomedica Ltd|Dual-virus vectors|

---

GB9907461D0|1999-03-31|1999-05-26|King S College London|Neurite regeneration|

---

GB0024550D0|2000-10-06|2000-11-22|Oxford Biomedica Ltd|

---

CN1620508A|2001-12-21|2005-05-25|牛津生物医学（英国）有限公司|Transgenic organism|

---

GB0325379D0|2003-10-30|2003-12-03|Oxford Biomedica Ltd|Vectors|

---

GB0526211D0|2005-12-22|2006-02-01|Oxford Biomedica Ltd|Viral vectors|

---

US7838729B2|2007-02-26|2010-11-23|Monsanto Technology Llc|Chloroplast transit peptides for efficient targeting of DMO and uses thereof|

---

US7806113B2|2008-02-07|2010-10-05|Jay Edward Skilling|Compressed gas projectile accelerator having multiple projectile velocity settings|

---

EP2575894B1|2010-05-28|2015-02-25|Oxford Biomedica Ltd|Delivery of lentiviral vectors to the brain|

---

US20120295960A1|2011-05-20|2012-11-22|Oxford Biomedica Ltd.|Treatment regimen for parkinson's disease|

---

EP2958996B1|2013-02-25|2019-10-16|Sangamo Therapeutics, Inc.|Methods and compositions for enhancing nuclease-mediated gene disruption|

---

JP2016521561A|2013-06-14|2016-07-25|セレクティス|A method for non-transgenic genome editing in plants|

---

SG10201800111SA|2013-07-09|2018-02-27|Harvard College|Multiplex rna-guided genome engineering|

---

EP3110945B1|2014-02-27|2021-09-08|Monsanto Technology LLC|Compositions and methods for site directed genomic modification|

---

CA2951707A1|2014-06-10|2015-12-17|Massachusetts Institute Of Technology|Method for gene editing|

---

CN113789317A|2014-08-06|2021-12-14|基因工具股份有限公司|Gene editing using Campylobacter jejuni CRISPR/CAS system-derived RNA-guided engineered nucleases|

---

WO2016065364A1|2014-10-24|2016-04-28|Life Technologies Corporation|Compositions and methods for enhancing homologous recombination|

---

GB201504223D0|2015-03-12|2015-04-29|Genome Res Ltd|Biallelic genetic modification|

---

US9790490B2|2015-06-18|2017-10-17|The Broad Institute Inc.|CRISPR enzymes and systems|

---

WO2017040511A1|2015-08-31|2017-03-09|Agilent Technologies, Inc.|Compounds and methods for crispr/cas-based genome editing by homologous recombination|

---

WO2017064546A1|2015-09-24|2017-04-20|Crispr Therapeutics Ag|Novel family of rna-programmable endonucleases and their uses in genome editing and other applications|

---

EP3443088A1|2016-04-13|2019-02-20|Editas Medicine, Inc.|Grna fusion molecules, gene editing systems, and methods of use thereof|

---

US20190309290A1|2016-04-29|2019-10-10|Basf Plant Science Company Gmbh|Improved Methods for Modification of Target Nucleic Acids|EP2734621B1|2011-07-22|2019-09-04|President and Fellows of Harvard College|Evaluation and improvement of nuclease cleavage specificity|

---

US10704021B2|2012-03-15|2020-07-07|Flodesign Sonics, Inc.|Acoustic perfusion devices|

---

US20150044192A1|2013-08-09|2015-02-12|President And Fellows Of Harvard College|Methods for identifying a target site of a cas9 nuclease|

---

US9359599B2|2013-08-22|2016-06-07|President And Fellows Of Harvard College|Engineered transcription activator-like effectordomains and uses thereof|

---

US9340799B2|2013-09-06|2016-05-17|President And Fellows Of Harvard College|MRNA-sensing switchable gRNAs|

---

US9322037B2|2013-09-06|2016-04-26|President And Fellows Of Harvard College|Cas9-FokI fusion proteins and uses thereof|

---

US9526784B2|2013-09-06|2016-12-27|President And Fellows Of Harvard College|Delivery system for functional nucleases|

---

US9068179B1|2013-12-12|2015-06-30|President And Fellows Of Harvard College|Methods for correcting presenilin point mutations|

---

US9725710B2|2014-01-08|2017-08-08|Flodesign Sonics, Inc.|Acoustophoresis device with dual acoustophoretic chamber|

---

AU2015298571B2|2014-07-30|2020-09-03|President And Fellows Of Harvard College|Cas9 proteins including ligand-dependent inteins|

---

CN108513575A|2015-10-23|2018-09-07|哈佛大学的校长及成员们|Nucleobase editing machine and application thereof|

---

US11236313B2|2016-04-13|2022-02-01|Editas Medicine, Inc.|Cas9 fusion molecules, gene editing systems, and methods of use thereof|

---

US11214789B2|2016-05-03|2022-01-04|Flodesign Sonics, Inc.|Concentration and washing of particles with acoustics|

---

KR20190034628A|2016-08-03|2019-04-02|프레지던트 앤드 펠로우즈 오브 하바드 칼리지|Adenosine nucleobase editing agent and uses thereof|

---

US10745677B2|2016-12-23|2020-08-18|President And Fellows Of Harvard College|Editing of CCR5 receptor gene to protect against HIV infection|

---

US11268082B2|2017-03-23|2022-03-08|President And Fellows Of Harvard College|Nucleobase editors comprising nucleic acid programmable DNA binding proteins|

---

KR20200089334A|2017-12-14|2020-07-24|프로디자인 소닉스, 인크.|Acoustic transducer driver and controller|

---

GB201913898D0|2019-09-26|2019-11-13|Lightbio Ltd|Nucleic acid construct|

法律状态:
2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

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US201762451859P| true| 2017-01-30|2017-01-30|

---

US62/451,859|2017-01-30|

---

PCT/EP2018/052313|WO2018138385A1|2017-01-30|2018-01-30|Repair template linkage to endonucleases for genome engineering|

---