use of cas protein, method for detecting nucleic acid molecules, and kit

专利摘要:
The present invention provides the use of a Cas protein, a method for detecting a target nucleic acid molecule and a kit. The method for detecting a target nucleic acid molecule includes adding a guide RNA, Cas12a and a nucleic acid probe to a reaction system containing a target nucleic acid molecule to be detected and detecting the nucleic acid probe aftercompletion of the reaction.
公开号:BR112020000809A2
申请号:R112020000809-5
申请日:2018-04-12
公开日:2020-09-08
发明作者:Jin Wang；Qiuxiang Cheng；Shiyuan Li；Xiaoyan Li；Linxian Li
申请人:Shanghai Tolo Biotechnology Company Limited；
IPC主号:

专利说明:

[0001] [0001] The present invention belongs to the field of biotechnology and, in particular, to a method for detecting a target nucleic acid molecule. BACKGROUND OF THE INVENTION
[0002] [0002] A specific nucleic acid detection method has an important application value, such as pathogen detection, genetic disease detection, etc. In one aspect of pathogen detection, since each pathogenic microorganism has its own unique molecular nucleic acid sequence, it is possible to develop the molecular detection of nucleic acids for specific species, also known as nucleic acid diagnosis (NAD), which is of great importance in the areas of food security, detection of pollution by environmental microorganisms, infection by human pathogens, etc. Another aspect is the detection of single nucleotide polymorphism (SNP) in humans or other species. Understanding the relationship between genetic variation and biological function at the genomic level provides a new perspective for modern molecular biology. The SNP is closely related to biological functions, evolution and disease; therefore, the development of SNP detection and analysis technologies is particularly important.
[0003] [0003] Currently, many NAD methods have been established, mainly for the detection of a specific DNA molecule, and there are also some methods for RNA molecules. In general, a DNA molecule is very stable; therefore, a test sample can come from a series of complex biological samples; because RNA degrades very easily, so it needs to be handled very carefully. In the 1970s, a detection method was established using restriction endonuclease digestion. Subsequently, methods such as Southern, Northern and dot blot hybridization were developed for specific detection of a nucleic acid molecule. In 1985, when PCR became a conventional experimental method, it led to an exponential improvement in molecular biology. The detection of a specific nucleic acid molecule currently established generally needs to be carried out in two stages, the first stage being the amplification of a target nucleic acid and the second stage the detection of the target nucleic acid. PCR technology is an amplification method that is first established and most commonly used today. Currently, based on the PCR method, a fluorescence-labeled probe is introduced, so that the amplification situation of a target can be detected in real time, called real-time PCR. Real-time PCR is not only a fast and highly sensitive detection method, but also a method for quantitative analysis. In addition to the PCR amplification method, many alternative methods have been established, such as ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, Loop-mediated isothermal amplification (LAMP), rolling-circle amplification (RCA ), Amplification of Polymerase Recombinase (RPA), etc. The advantage of many of these alternative methods is isothermality. That is, only one temperature is needed to complete the reaction, without the need for a thermal cycling instrument such as that used in PCR. Among nucleic acid detection methods, in addition to real-time PCR that can directly complete amplification and detection, FISH technology (fluorescence in situ hybridization) is the most commonly used detection method - a method in which a molecular probe labeled is hybridized in situ to a complementary target sequence. In addition, detection methods such as state-of-the-art sequencing technologies and Oxford Nanopore sequencing technologies have also been developed, but these methods often require expensive experimental equipment.
[0004] [0004] The detection of SNPs also requires first amplification by a method such as PCR and the like, in order to obtain enough region fragments containing SNP sites for further detection. Commonly used methods include: primer extension, hybridization, ligation and enzymatic cleavage. When the above methods are completed, a specific method needs to be used for detection, such as mass spectrometry detection, fluorescence detection, chemiluminescence detection, etc.
[0005] [0005] Although many detection methods have been developed for the detection of nucleic acids, as described above, in certain cases, how to detect more quickly, simply and economically is still an important development direction, such as the rapid detection of pathogenic bacteria in the field, rapid detection of drug-sensitive SNPs, etc. In 2016, Collins et al. developed a fast and inexpensive method to detect the Zika virus based on the characteristic of CRISPR-Cas9 to specifically recognize and cleave a target sequence. In 2017, Feng Zhang et al. established a rapid method of nucleic acid detection using a "side effect" feature of CRISPR-Cas13a. The "side effect" means that Cas13a binds to a specific target RNA and randomly cleaves other non-target RNAs (here RNA molecules are designed as an RNA fluorescence notification system); Rapid detection of target RNA is performed in combination with an RPA isothermal amplification technology, and Feng Zhang's team named this detection method SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking; Specific High Sensitivity Enzymatic Reporter UnLOCKing in English ). The SHERLOCK method involves binding to an RNA model; therefore, if DNA detection is required, the DNA must first be transcribed into an RNA model for detection; and given the instability of the RNA, this method will undoubtedly increase the degree of difficulty of operation.
[0006] [0006] In 2015, Feng Zhang et al. discovered a new CRISPR-related Cas12a endoproteinase (formerly known as Cpf1), which, like a commonly used Cas9 protein, is a specific RNA-guided DNA endonuclease; however, compared to Cas9, Cas12a has its own characteristics, for example, only one crRNA is needed to guide the specific cleavage of a double-stranded DNA and a sticky end is produced. SUMMARY
[0007] [0007] An object of the present invention is to provide a method for detecting a target nucleic acid molecule.
[0008] [0008] Another objective of the present invention is to provide the use of a Cas protein in a method for detecting a target nucleic acid molecule.
[0009] [0009] In a first aspect of the present invention, a kit is provided that includes a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution.
[0010] [0010] A method for detecting a target nucleic acid molecule includes adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution in a reaction system containing a target nucleic acid molecule to be detected and, in then, detect the target nucleic acid (especially by a fluorescence intensity detection method).
[0011] [0011] Preferably, the Cas protein is Cas12a or Cas protein with a single-stranded DNA cleavage side activity similar to that of Cas12a.
[0012] [0012] Preferably, the Cas protein is Cas12a.
[0013] [0013] Cas12a is preferably one among FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.
[0014] [0014] Preferably, Cas12a is LbCas12a.
[0015] [0015] Preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind to a target DNA.
[0016] [0016] In another preferred embodiment, the nucleic acid probe is single-stranded DNA; single-stranded DNA is preferably fluorescently labeled single-stranded DNA; single-stranded DNA is preferably a fluorescent probe that is labeled with a HEX fluorescent group at a 5 'terminus and labeled with a BHQ1 suppressor group at a 3' terminus.
[0017] [0017] In another preferred embodiment, the method for detecting the nucleic acid probe is preferably a fluorescence detection method; and the fluorescence detection method is preferably a detection method that uses a microplate reader or a fluorescence spectrophotometer.
[0018] [0018] Preferably, the target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained after amplification.
[0019] [0019] Preferably, the detection method of the present invention can be used to detect a pathogenic microorganism, genetic mutation or a specific target DNA.
[0020] [0020] In another preferred embodiment, the Cas protein includes Cas12b (C2c1).
[0021] [0021] In a second aspect of the present invention, use of a Cas protein is provided in a method for detecting a target nucleic acid molecule or in the preparation of a formulation for detecting a target nucleic acid molecule.
[0022] [0022] In another preferred embodiment, when a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system.
[0023] [0023] Preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind to a target DNA.
[0024] [0024] In a third aspect of the present invention, a kit is provided that includes a guide RNA, a Cas protein and a nucleic acid probe.
[0025] [0025] In another preferred embodiment, the kit also includes a buffer solution.
[0026] [0026] In a fourth aspect of the present invention, a detection system is provided to detect a target nucleic acid molecule, wherein the system includes: (a) a Cas protein, which is Cas12a or a Cas protein with an activity of cleavage of single-stranded DNA similar to that of Cas12a; (b) a guide RNA that guides the Cas protein to specifically bind to the target nucleic acid molecule; and (c) a nucleic acid probe that is single-stranded DNA; wherein the target nucleic acid molecule is target DNA.
[0027] [0027] In another preferred embodiment, the detection system also includes (d) a buffer solution.
[0028] [0028] In another preferred embodiment, the detection system also includes a target nucleic acid molecule to be detected.
[0029] [0029] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 1-100 copies / microliter or 1015 copies / microliter, preferably 1-10 copies / microliter, and more preferably 1- 5 copies / microliter.
[0030] [0030] In another preferred embodiment, in the detection system, the molar ratio of the nucleic acid probe to the target nucleic acid molecule is 10 3: 1 to 1014: 1 and, preferably, 104: 1 to 107: 1.
[0031] [0031] In another preferred embodiment, the target nucleic acid molecule detection site is located at positions 1-12 downstream of the
[0032] [0032] In another preferred embodiment, the length of the guide RNA is 15-30 nt and, preferably, 15-18 nt.
[0033] [0033] In another preferred embodiment, the target DNA includes a cDNA.
[0034] [0034] In another preferred embodiment, the target DNA is selected from a group consisting of single-stranded DNA, double-stranded DNA or a combination thereof.
[0035] [0035] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a suppressor group.
[0036] [0036] In another preferred embodiment, the fluorescent group and the suppressor group are located independently at the 5 'terminal, the 3' terminal and the middle portion of the nucleic acid probe.
[0037] [0037] In another preferred embodiment, the length of the nucleic acid probe is 3-300 nt, preferably 5-100 nt, more preferably 6-50 nt, and even more preferably 8-20 nt.
[0038] [0038] In another preferred embodiment, the target nucleic acid molecule includes a target nucleic acid molecule derived from a group consisting of plants, animals, insects, microorganisms, viruses or a combination thereof.
[0039] [0039] In another preferred embodiment, the target DNA is artificially synthesized or naturally occurring DNA.
[0040] [0040] In another preferred embodiment, the target DNA includes wild-type or mutant DNA.
[0041] [0041] In another preferred embodiment, the target DNA includes DNA obtained by reverse RNA transcription or amplification, such as a cDNA etc.
[0042] [0042] In another preferred modality, Cas12a is selected from a group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; and most preferably, Cas12a is LbCas12a.
[0043] [0043] In another preferred embodiment, the Cas protein with a single-stranded DNA strand cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (i.e., C2c1).
[0044] [0044] In another preferred embodiment, the Cas12b protein is selected from a group consisting of AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus), AkCas12b (Alicyclobacillus kakegawensis), AmCas12b (Alicyclobusillacillus , and AcCas12b (Alicyclobacillus contaminans).
[0045] [0045] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA that carries a detectable marker.
[0046] [0046] In another preferred embodiment, single-stranded DNA is single-stranded DNA fluorescently and biotin-tagged.
[0047] [0047] In another preferred embodiment, single-stranded DNA is single-stranded DNA fluorescently labeled.
[0048] [0048] In another preferred embodiment, single-stranded DNA is a fluorescent probe that is labeled with a HEX fluorescent group at a 5 'terminal and is labeled with a BHQ1 suppressor group at a 3' terminal.
[0049] [0049] In a fifth aspect of the present invention, a kit is provided to detect a target nucleic acid molecule, wherein the kit includes: i) a first container and a Cas protein in the first container, the Cas protein being Cas12a or protein Cas having collateral single-stranded DNA cleavage activity similar to Cas12a; ii) an optional second container and a guide RNA in the second container, the guide RNA guiding the Cas protein to specifically bind to the target nucleic acid molecule; iii) a third container and a nucleic acid probe in the third container; and iv) an optional fourth container and a buffer solution located in the fourth container; wherein the target nucleic acid molecule is target DNA.
[0050] [0050] In another preferred embodiment, any two, three or four (or all) of the first, second, third and fourth containers can be the same or different containers.
[0051] [0051] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a suppressor group.
[0052] [0052] In a sixth aspect of the present invention, a method is provided to detect whether a target nucleic acid molecule exists in a sample, including the following steps:
[0053] [0053] (a) providing the detection system to detect a target nucleic acid molecule according to the fourth aspect of the present invention, wherein the detection system still has a sample to be detected; and
[0054] [0054] (b) detecting whether the nucleic acid probe in the detection system is cleaved by a Cas protein, wherein the cleavage is a transcleavage of a single-stranded collateral DNA;
[0055] [0055] wherein if the nucleic acid probe is cleaved by the Cas protein, then this indicates the presence of the target nucleic acid molecule in the sample; and if the nucleic acid probe is not cleaved by the Cas protein, this indicates the absence of the target nucleic acid molecule in the sample.
[0056] [0056] In another preferred embodiment, the sample to be detected includes a non-amplified sample and an amplified sample (or amplified by nucleic acid).
[0057] [0057] In another preferred modality, the sample to be detected is a sample obtained by amplification.
[0058] [0058] In another preferred embodiment, a method for amplifying nucleic acid is selected from a group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, mediated amplification by transcription, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
[0059] [0059] In another preferred embodiment, PCR includes high temperature PCR, normal temperature PCR and low temperature PCR.
[0060] [0060] In another preferred embodiment, the method is used to detect whether SNP, point mutation, deletion and / or insertion exist in a nucleic acid at a target site.
[0061] [0061] In another preferred modality, when the upstream and downstream regions (in the range of -20 nt to +20 nt, preferably in the range of - 15 nt to +15 nt, and more preferably in the range of -10 nt to +10 nt) of a target site do not have a PAM sequence, nucleic acid amplification is performed using a PAM introducer primer.
[0062] [0062] In another preferred embodiment, the PAM introducer primer has a structure of formula I in 5'-3 ':
[0063] [0063] P1-P2-P3 (I)
[0064] [0064] where,
[0065] [0065] P1 is a 5 'segment sequence located at the 5' end and is complementary or not complementary to the sequence of the target nucleic acid molecule;
[0066] [0066] P2 is a PAM sequence;
[0067] [0067] P3 is a 3 'segment sequence located at the 3' terminal and is complementary to the sequence of the target nucleic acid molecule.
[0068] [0068] In another preferred embodiment, the PAM primer specifically binds upstream or downstream of the target nucleic acid molecule.
[0069] [0069] In another preferred modality, P1 has a length of 0-20 nt.
[0070] [0070] In another preferred modality, P3 has a length of 5-20 nt.
[0071] [0071] In another preferred embodiment, the PAM primer has a length of 18-50 nt, and preferably 20-35 nt.
[0072] [0072] In another preferred modality, the complementation includes complete and partial complementation.
[0073] [0073] In another preferred embodiment, at least one primer containing the PAM sequence is used in nucleic acid amplification.
[0074] [0074] In another preferred modality, when the upstream and downstream region (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, and more preferably in the range of -10 nt to +10 nt) of the target site contains a PAM sequence, a primer containing or not the PAM sequence can be used and the amplified amplification product contains the PAM sequence.
[0075] [0075] In another preferred embodiment, the detection in step (b) includes a fluorescence detection method.
[0076] [0076] In another preferred embodiment, the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer for detection.
[0077] [0077] In a seventh aspect of the present invention, use of a Cas protein is provided in the preparation of a reagent or detection kit to detect a target nucleic acid molecule based on single-stranded DNA strand cleavage, wherein the protein Cas is a Cas12a or Cas protein with a collateral single-stranded DNA cleavage activity similar to that of Cas12a.
[0078] [0078] In another preferred modality, Cas12a is selected from a group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; and most preferably, Cas12a is LbCas12a.
[0079] [0079] In another preferred embodiment, the Cas protein with a single-stranded DNA collateral cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (or C2c1).
[0080] [0080] In another preferred embodiment, the Cas12b protein is selected from a group consisting of AacCas12b.
[0081] [0081] It should be understood that, within the scope of the present invention, the aforementioned technical characteristics of the present invention and the technical characteristics described in detail below (for example, in examples) can be combined together to form a new technical solution or preferential. Due to the length limit, it will not be repeated here. BRIEF DESCRIPTION OF THE FIGURES
[0082] [0082] FIGURE 1 shows a cis cleavage characteristic of Cas12a in the cleavage of a single stranded target DNA.
[0083] [0083] FIGURE 2 shows that when cleaving a single stranded target DNA, Cas12a does not depend on a PAM sequence necessary for cleaving double strands.
[0084] [0084] FIGURE 3 shows a Cas12a transcleavage characteristic in the cleavage of a single stranded target DNA.
[0085] [0085] FIGURE 4 shows test Cas12as from 10 different sources, where all of these Cas12as have cis-cleavage and trans-cleavage activities in a single-stranded DNA.
[0086] [0086] FIGURE 5 identifies sites possibly related to cis cleavage and transcleavage activities in single stranded DNA in Cas12a through a Cas12a single site mutation experiment.
[0087] [0087] FIGURE 6 shows the structures of the monomers of Cas12a and
[0088] [0088] FIGURE 7 shows the fluorescence values obtained by different Cas12as using a specific double-stranded DNA substrate and a single-stranded DNA (HEX-N12-BHQ1) as a fluorescence detection probe. The negative control group is not added with the specific substrate.
[0089] [0089] FIGURE 8 shows a schematic flowchart of a HOLMES method for detecting a target DNA based on the amplification of the target DNA and the Cas12a transcleavage activity in a single-stranded DNA.
[0090] [0090] FIGURE 9 shows a sensitivity test of a target DNA using FnCas12a or LbCas12a directly, or in combination with the HOLMES method.
[0091] [0091] FIGURE 10 shows fluorescence detection values of target sequences with different single-point mutations, as detected by the HOLMES method, using crRNAs of different lengths of guide sequences in combination with FnCas12a or LbCas12a.
[0092] [0092] FIGURE 11 tests whether a single-stranded DNA probe labeled with FAM is transcleaved after the addition of single-stranded target DNA using a fluorescent probe labeled with FAM and 10 Cas12a proteins.
[0093] [0093] FIGURE 12 tests fluorescence values after adding the single stranded target DNA using HEX-N12-BHQ1 as a probe and 10 Cas12a proteins.
[0094] [0094] FIGURE 13 (A) shows the detection values of HOLMES when a fragment of the gyrB gene is used as a target sequence and different concentrations of pure culture Escherichia coli MG1655 are used as positive control models using a fluorescent probe. Single-stranded DNA marked with HEX and BHQ1 at two ends of the same. It is shown that the value of the fluorescence response of Escherichia coli MG1655 decreases with the decrease of its concentration. (B) Detection values of water samples in environments in different locations.
[0095] [0095] FIGURE 14 shows a schematic flowchart of a HOLMES method for detecting SNP and fluorescence detection values for 5 SNP sites.
[0096] [0096] FIGURE 15 shows the fluorescence detection values of key sites in a TP53 gene (a cancer-related gene), as detected by the HOLMES method.
[0097] [0097] FIGURE 16 shows the detection values of 5 SNP sites (related to gout), as detected by the HOLMES method.
[0098] [0098] FIGURE 17 shows the detection values of a SNP site (related to gout), as detected by the HOLMES method, in which the samples are samples from 21 volunteers.
[0099] [0099] FIGURE 18 shows a primer design scheme of an example of the present invention, which can be used for the detection of SNP by HOLMES anywhere.
[0100] [0100] FIGURE 19 uses a combination of LAMP and HOLMES to detect Escherichia coli in the system. (A) An electrophoresis map of a LAMP amplified Escherichia coli gyrB gene. A total of two sets of primers gyrB-1 and gyrB-2 are used for amplification. gyrB is the characteristic gene of Escherichia coli. (B) A HOLMES detection system is used to detect an LAMP amplification product. Negative control: the sample is sterile water and a gyrB-1 amplification primer is used to amplify or detect the result of the gyrB gene; gyrB-1: the sample is Escherichia coli to be detected and a first set of primers for amplifying the gyrB gene is used to amplify or detect the result of the gyrB gene; and gyrB-2: the sample is Escherichia coli to be detected, and a second set of gyrB gene amplification primers is used to amplify or detect the gyrB gene result.
[0101] [0101] FIGURE 20 detects the genotype of a human HEK293T cell using a combination of LAMP and HOLMES. (A) An electrophoresis map of a corresponding SNP detection model of the human HEK293T cell, as amplified by LAMP. Negative control: the sample is sterile water and the result is the result of the amplification using an rs5082 amplification primer; rs5082: the sample is a total genome of the human HEK293T cell and the result is the result of amplification using the rs5082 amplification primer; and rs1467558: the sample is the total human cell genome HEK293T and the result is the result of amplification using an rs1467558 amplification primer. (B) A HOLMES detection system is used to detect a LAMP amplification product. The rs5082 site was detected using two crRNA-G and crRNA-T crRNAs, respectively (Sequence Listing 5); and the rs1467558 site was detected using two crRNA-C and crRNA-T crRNAs, respectively, (Sequence Listing 5).
[0102] [0102] FIGURE 21 uses a combination of RPA and HOLMES to detect Escherichia coli in the system. (A) Amplification of the Escherichia coli gyrB gene by RPA. A total of two sets of primers gyrB-1 and gyrB-2 are used for amplification. gyrB is a characteristic gene of Escherichia coli. (B) A HOLMES detection system is used to detect an RPA amplification product. Negative control: the sample is sterile water and a gyrB-1 amplification primer is used to amplify or detect the result of the gyrB gene; gyrB-1: the sample is Escherichia coli to be detected and a first set of gyrB amplification primers is used to amplify or detect the result of the gyrB gene; and gyrB-2: the sample is Escherichia coli to be detected, and a second set of gyrB amplification primers is used to amplify or detect the result of the gyrB gene.
[0103] [0103] FIGURE 22 shows the detection of Cas12b collateral single-stranded DNA cleavage activity when single-stranded DNA is used as the target DNA. After the completion of the collateral cleavage reaction, the reagents are separated by electrophoresis on 12% urea denatured gel and detected by a fluorescence imaging system. The numbers in parentheses represent the final concentrations of the reagents in nM; the target DNA is a single-stranded DNA of 66 nt in length at a dosage of 50 nM; and the single-stranded DNA probe is single-stranded DNA that carries a FAM marker at the 5 'end at a dosage of 50 nM. As can be seen in the figure, after Cas12b, the guide RNA and target DNA are contained, the single-stranded DNA marked with FAM is cut into fragments, that is, Cas12b has a single-stranded DNA cleavage activity.
[0104] [0104] FIGURE 23 shows the detection of Cas12b collateral single stranded DNA cleavage activity when single stranded DNA and double stranded DNA are used as target DNAs. Upon completion of the cleavage reaction, the reagents are detected using a fluorescence microplate reader. Both Cas12b and guide RNA dosages are 500 nM; the target DNA is a single-stranded DNA of 66 nt in length or double-stranded DNA at a dosage of 50 nM; and the single-stranded DNA probe is a single-stranded DNA probe (HEX-N12-BHQ1) containing a fluorescence reporter group and a suppressor group at the dosage of 500 nM. As can be seen in the figure, regardless of whether a single-stranded DNA model or a double-stranded DNA model is used, collateral single-stranded DNA cleavage activity can be detected after the addition of Cas12b and the guide RNA.
[0105] [0105] FIGURE 24 shows the transcleavage activity of Cas12b collateral single-stranded DNA into a low concentration target DNA after combination with LAMP amplification. DETAILED DESCRIPTION
[0106] [0106] In order to clarify the objectives, technical solutions and advantages of the modalities of the present invention, the following clearly and completely describes the technical solutions in the modalities of the present invention with reference to the drawings accompanying the modalities of the present invention. The described modalities are a part, and not all, of the present invention. All other modalities obtained by a person of ordinary skill in the art based on the modalities of the present invention without creative efforts fall within the scope of protection of the present invention.
[0107] [0107] The inventor has developed a technical solution for the detection of target nucleic acid through extensive and in-depth research and research on the cleavage characteristics of a Cas enzyme (such as Cas12a and Cas12b enzymes). The experimental results show that a nucleic acid is detected successfully and quickly when using the aforementioned technical solution, for example, to identify whether there is a certain concentration of microorganisms like Escherichia coli in the water and to quickly identify a SNP genotype. The present invention is concluded on the basis of this. Terms
[0108] [0108] The term "guide RNA" refers to an RNA that guides a Cas protein to specifically bind to a target DNA sequence.
[0109] [0109] The term "crRNA" refers to a CRISPR RNA, which is a short RNA that guides Cas12a to bind to a target DNA sequence.
[0110] [0110] The term "CRISPR" refers to a short palindromic repetition regularly interspersed together, which is the immune system of many prokaryotes.
[0111] [0111] The term "Cas protein" refers to a protein associated with
[0112] [0112] The term "Cas12a" (formerly called "Cpf1") refers to a crRNA-dependent endonuclease, which is a V-A type enzyme in the CRISPR system classification.
[0113] [0113] The terms "Cas12b" and "C2c1" are used interchangeably and refer to a crRNA-dependent endonuclease, which is a V-B type enzyme in the CRISPR system classification.
[0114] [0114] The term "LAMP" is a loop-mediated isothermal amplification technology, which is a nucleic acid isothermal amplification technology suitable for the diagnosis of genes.
[0115] [0115] The term "PAM" refers to a motif adjacent to the protospace, necessary for the cleavage of Cas12a. The Pn of FnCas12a is a TTN sequence, the PAM of LbCas12a is a TTTN sequence and the PAM of AacCas12b is TTN.
[0116] [0116] The present invention discloses a method for detecting a target nucleic acid molecule, which includes: adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution in a reaction system containing a nucleic acid molecule target to be detected and then perform fluorescence detection of the target nucleic acid molecule.
[0117] [0117] The Cas protein is Cas12a or Cas12b.
[0118] [0118] Cas12a is preferably one among FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; and Cas12a is preferably LbCas12a.
[0119] [0119] Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b or AcCas12b.
[0120] [0120] Guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.
[0121] [0121] The target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained by amplification.
[0122] [0122] The detection method can detect a pathogenic microorganism, genetic mutation or a specific target DNA.
[0123] [0123] Use of a Cas protein in a method to detect a target nucleic acid molecule.
[0124] [0124] When a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system.
[0125] [0125] Guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.
[0126] [0126] The present invention also provides a kit that includes a guide RNA, a Cas protein and a nucleic acid probe. In addition, the kit of the present invention can also include a buffer solution.
[0127] [0127] The present invention provides a detection method for quickly detecting a target nucleic acid molecule with high specificity. When target DNA (single-stranded or double-stranded), crRNA and Cas12a protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system. Through the project, the crRNA is directed to the target DNA (a segment of the DNA sequence to be detected); crRNA and Cas12a protein are added to the detection system; when target DNA is present, Cas12a forms a ternary complex with the crRNA and target DNA, and in the meantime, the complex exerts its collateral cleavage activity and cleaves a single-stranded DNA marked with a fluorescent signal (two ends of the DNA single-stranded cells are respectively connected to a luminescent group and a suppressor group, and the luminescent group can emit light after being cleaved), thus emitting fluorescence. Therefore, it can be determined whether the system to be detected contains the target DNA molecule through fluorescence detection. Using the method of the present invention, it is possible to quickly detect whether a specific DNA sequence is contained in a sample. The sensitivity of the detection method can be greatly improved by combining it with PCR technology. The nucleic acid probe of the present invention is preferably a fluorescent probe. HOLMES condition test:
[0128] [0128] The present invention provides the use of Cas12 enzymes, such as Cas12a and Cas12b, in the detection of nucleic acids. The following description uses Cas12a as an example.
[0129] [0129] Selection of Cas12a: according to the research, Cas12a has a transcleavage activity, that is, since the target DNA, the crRNA and the Cas12a protein form a ternary complex, other single-stranded DNAs (strand DNAs collateral) in the system will be cleaved. According to this principle, a specific DNA detection method is designed. First, the collateral DNA is designed as a fluorescent probe, consisting of a 12 nt random sequence, and is labeled with a HEX fluorescent group at the 5 'terminal and a BHQ1 (HEX-N12-BHQ1) suppressor group at the 3' terminal . When the target DNA fragment is contained in the system, a ternary complex of the target DNA, crRNA and the Cas12a protein will be formed. At that time, the probe will be cleaved and, in the meantime, the HEX fluorescent group will emit fluorescence (with an excitation light at 535 nM and an emission light at 556 nM), as detected by a fluorescence detector. Next, 10 different Cas12as are tested and the target sequence is double-stranded DNA, as shown in FIGURE 7. It is possible to observe that the complex composed of the double-stranded target DNA and each Cas12a protein can perform the transcleavage activity.
[0130] [0130] HOLMES response sensitivity: then, the response sensitivities of FnCas12a and LbCas12a to the target DNA are tested, that is, the lowest concentration of the target DNA in which the response can occur is investigated. As shown in FIGURE 9, when the test target is added directly, they can respond to the target DNA with a concentration above 0.1 nM, and the response is noticeable when the concentration is above 1 nM. If a PCR technology (the HOLMES method) is combined, as shown in FIGURE 8, that is, amplification of the fragment of interest by PCR followed by a Cas12a cleavage reaction, the sensitivity of the response may be as low as 10 aM , as shown in FIGURE 9.
[0131] [0131] SNP test: next, it is tested whether the HOLMES method can detect a SNP genotype. T1 is used as a target sequence, PAM at this site is mutated, or positions 1-18 of the target sequence are subject to single-point mutation, respectively, and the detection differences between an unmuted and a mutated sequence by different length crRNAs are compared.
[0132] [0132] As shown in FIGURE 10, when the target complementary sequence is a 24 nt crRNA (crRNA-24nt), the single point mutation at the positions
[0133] [0133] In the present invention, Cas12a cleaves single-stranded DNA through a programmed form of cleavage independent of the PAM sequence, which is called cis cleavage; but once the target Cas12a / crRNA / DNA ternary complex is formed, it will exhibit a transcleavage activity, that is, it will exhibit a cleavage activity of any single non-target strand DNA in the system.
[0134] [0134] Based on the characteristics of Cas12a, a method for specific detection of nucleic acid molecules, called HOLMES (a Simple, Efficient, Low Cost, Multipurpose, one-hour test; one Hour Low-cost Multipurpose Efficient Simple in English). As the name says, the technology is characterized by a fast speed test method (1 hour), low price, multiple channels, high efficiency and simple. The method can be used in the fields of rapid pathogen detection, SNP detection and the like. Nucleic acid detection based on collateral cleavage activity
[0135] [0135] The present invention also provides a method of detecting nucleic acid based on the collateral cleavage activity of the Cas12 enzyme (including Cas12a or Cas12b).
[0136] [0136] Preferably, the detection of the present invention can be performed for SNP and, in particular, PCR amplification is performed first,
[0137] [0137] With reference to FIGURE 18, a primer design scheme is provided.
[0138] [0138] Case 1. When there is a PAM site close to the SNP site, a crRNA synthesized based on a guide sequence designed according to the PAM site can be used for HOLMES detection. When the HOLMES method is used for detection, it shows a relatively low background signal; and for the same guide sequence, the signal differences between the different SNP models are quite large.
[0139] [0139] Case 2. When there is no PAM site or a suitable PAM site close to the SNP site, a PAM site can be introduced according to the experimental scheme above.
[0140] [0140] A typical step includes designing a primer close to the SNP site and transporting a PAM site on the primer, where the sequence located at the 3 'terminal of the PAM site must be paired with the model DNA. There is no special requirement for the primer at the other end, as long as the primer can be paired with the model DNA and can be used for PCR amplification. As shown in FIGURE 18, PAM sites can be successfully introduced after PCR amplification.
[0141] [0141] With reference to FIGURE 10, in the present invention, when designing the introduction of the PAM site, the SNP site is usually located in the first 16 bases of the 5 'terminal of the crRNA guide sequence, preferably at positions 1-14, more preferably in positions 1-12, even more preferably in positions 1-11 or 1-10, and much more preferably in positions 1-8 or 1-7.
[0142] [0142] The present invention has the following main advantages:
[0143] [0143] (1) Fast speed: when the test conditions are ready, it takes only 1 hour between obtaining the sample and obtaining the test results.
[0144] [0144] (2) Low cost: there are no special materials or enzymes in the experiment and it involves a small amount of materials and reagents and, therefore, can be used for trace analysis.
[0145] [0145] (3) High efficiency: the present invention has extremely high sensitivity and can detect DNA at a concentration of 10 amM.
[0146] [0146] (4) Multiple uses: can detect different nucleic acid samples, including DNA and RNA samples.
[0147] [0147] (5) Simplicity: there are no special or complicated steps and, if a kit is prepared and a program is defined, only simple operations, such as adding a sample, are needed.
[0148] [0148] The present invention will be described in detail below with respect to specific examples. It should be understood that the following examples are intended only to illustrate the present invention, rather than to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989 ), or those recommended by the manufacturers. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.
[0149] [0149] Unless specifically stated otherwise, the experimental materials involved in the present invention can be obtained from commercial channels. Materials
[0150] [0150] 1. RNase inhibitor is purchased from TaKaRa and KOD FX high fidelity DNA polymerase is purchased from ToYoBo; primers (oligonucleotides) are synthesized by Sangon Biotech (Shanghai) Co., Ltd .; RNA polymerase T7 is purchased from Thermo; the RNA purification and concentration kit (RNA Clean & ConcentratorTM_5) is purchased from Zymo Research; the Wizard® SV gel and PCR cleaning system is purchased from Promega; and all media (eg, tryptone, yeast extract, etc.) are purchased from OXOID.
[0151] [0151] 2. Medium formula: a liquid LB (1% tryptone, 0.5% yeast extract, 1% NaCl) and only 2% agar need to be added to the liquid LB when a solid LB is prepared . Example 1 Cas12a detection protein capable of detecting single-stranded target DNA (probe being labeled with FAM)
[0152] [0152] A single-stranded DNA (target T1-R) was selected as a target sequence to test the response values of its detection by different Cas12a proteins.
[0153] [0153] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7-T1-24-R oligonucleotide, as shown in Table 5. Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and then cooling to 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermal cycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0154] [0154] 2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, a Cas12a (0.25 μM), a single-stranded target DNA (T1- Target R) (0.01 μM), a nucleic acid probe (N25-5 'FAM) (0.01 μM), a buffer solution of NEB buffer 3.1 and 0.5 μL of an RNase inhibitor. The blank control reaction was a reaction in which all other components were added, except the target single stranded DNA sequence. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0155] [0155] 3. Fluorescence detection: the reaction was subjected to urea-acrylamide gel electrophoresis (Urea-PAGE) and then detected with a fluorescence luminescence image generator. As shown in FIGURE 11, different Cas12s have different detection effects on the target. For example, for HkCas12a etc., the probe cleavage was caused even when no single target strand DNA was added. LbCas12a and the like are better candidates for Cas12a proteins because probe cleavage occurred only when single stranded target DNA was added. Example 2 detection of Cas12a protein capable of detecting single-stranded target DNA (probe being tagged with two HEX and BHQ1 markers)
[0156] [0156] A single-stranded DNA (target T1-R) was selected as the target sequence to test the response values of its detection by different Cas12a proteins.
[0157] [0157] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7-T1-24-R oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0158] [0158] 2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, a Cas12a (0.25 μM), a single-stranded target DNA (T1- Target R) (0.01 μM), a fluorescence probe (HEX-N12-BHQ1, that is, a single-stranded 12 nt DNA labeled with HEX at the 5 'terminal and with BHQ1 at the 3' terminal) (0, 5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The control reaction was a reaction in which all other components were added, except the target single stranded DNA sequence. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0159] [0159] 3. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 12, different Cas12s have different detection effects on the target. For example, for HkCas12a etc., the probe cleavage was caused even when no single target strand DNA was added. FnCas12a and the like are better candidates for Cas12a proteins because probe cleavage occurred only when single stranded target DNA was added. Example 3 detection of Cas12a protein capable of detecting target double-stranded DNA
[0160] [0160] A double-stranded DNA (target T1) was selected as a target sequence to test the response values of its detection by different proteins
[0161] [0161] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7-T1-24-R oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0162] [0162] 2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, a Cas12a (0.25 μM), a double-stranded target DNA (T1 target, obtained by annealing T1-F primers to target T1-R) (0.01 μM), a fluorescence probe (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0163] [0163] 3. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 7, different Cas12as have different detection effects on the target. LbCas12a and the like are better candidates for Cas12a proteins because probe cleavage occurred only when double stranded target DNA was added. Example 4 Testing of different concentrations of the target with FnCas12a and LbCas12a
[0164] [0164] the target T1 was selected as the target DNA and then diluted in different concentrations in a gradient to test the sensitivity of the response of FnCas12a and LbCas12a to it. To increase the sensitivity, a PCR amplification step was added.
[0165] [0165] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7- oligonucleotide
[0166] [0166] 2. PCR amplification (optional): a plasmid containing the target T1 target (pUC18-T1) was used as a model, diluted in a gradient and then used for the PCR reaction. The total volume of each reaction system was 20 μL, 0.25 μM M13F-47 and M13R-48 were used as primers (Table 4) and a KOD FX high fidelity enzyme (ToYoBo) was used for the PCR reaction . The PCR reaction procedure was at 95 ° C for 2 min and then started 35 cycles at 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. Upon completion of the PCR, the PCR amplification product was used directly for a Cas12a reaction.
[0167] [0167] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, FnCas12a or LbCas12a (0.25 μM), 1 μL of a PCR product ( or target DNAs that are directly diluted to different concentrations), a fluorescent probe (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0168] [0168] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 9, when the test target was added directly, all target DNAs with a concentration above 0.1 nM were able to respond, and the response was remarkable when the concentration was above 1 nM. If a PCR technology were combined, that is, amplification of the fragment of interest by means of PCR followed by a Cas12a cleavage reaction, the sensitivity of the response could be as low as 10 µM.
[0169] [0169] the target-T1 was selected as the target and was subjected to single point mutation in a PAM region and at positions 1-18, respectively, in order to test the response values of various crRNAs of different lengths for the wild type and the same after single point mutation.
[0170] [0170] 1. Preparation of crRNA: first, a transcription model was prepared by respective annealing of T7-crRNA-F with the synthesized oligonucleotides T7-T1-24-R, T7-T1-15-R, T7-T1- 16-R, T7-T1-17-R and T7-T1-18-R (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0171] [0171] 2. PCR amplification: a plasmid containing the target T1 target (pUC18-T1) was used as a model. The total volume of each reaction system was 20 μL, 0.25 μM of the M13R-48 primer and each respective target T1- F mutation primer was used (Table 4), and a KOD FX high fidelity enzyme (ToYoBo) was used for the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. After PCR completion, the product was used directly for a Cas12a reaction.
[0172] [0172] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, FnCas12a or LbCas12a (0.25 μM), 1 μL of a PCR product, a fluorescent probe (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0173] [0173] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 10, when the target complementary sequence was a 24 nt crRNA (crRNA-24nt), the single-point mutations at positions 8-18 were not very different from the wild type, while the fluorescence value obviously decreased after PAM mutation and the point mutation of positions 1-
[0174] [0174] The Escherichia coli gyrB gene was selected as a detection target to indirectly test the concentrations of Escherichia coli and similar microorganisms in water. Considering Escherichia coli MG1655 as a positive control, the content of microorganisms in the water (such as sewage and tap water) in the environment was determined.
[0175] [0175] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0176] [0176] 2. PCR amplification: when the positive control sample Escherichia coli MG1655 was grown until OD600 reached about 0.5, it was diluted by a gradient of 10 times, respectively, and then used as a model, and the sample was ambient water (including tap water and muddy water in the environment). The total volume of each reaction system was 20 μL, 0.25 μM of each of the gyrB-F and gyrB-R primers that were used (Table 4) and a KOD FX high fidelity enzyme (ToYoBo) was used for the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. After PCR completion, the PCR product was used directly for a Cas12a reaction.
[0177] [0177] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, LbCas12a (0.25 μM), 1 μL of a PCR product, a probe fluorescence (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0178] [0178] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 13, the fluorescence response value of Escherichia coli MG1655 decreases with decreasing concentration. Among them, microorganisms were most obviously detected in samples 2, 4, 5 and 6. Example 7 Human SNP test
[0179] [0179] The SNP test selected 5 human SNP sites, namely rs5082, rs1467558, rs2952768, rs4363657 and rs601338, to test the viability of the HOLMES method.
[0180] [0180] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C
[0181] [0181] 2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of each primer (table 4), 1 ng of a human genome (HEK293T) or directly scraped oral epithelial mucosa were used as a model, and a high fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. After PCR completion, the product was used directly for a Cas12a reaction. (Primers 1-rs5082-F-T, 2-rs1467558-F-T and 3-rs2952768-R-C were introduced directly into the corresponding SNP mutation products)
[0182] [0182] 3. Cas12a reaction: a 20 μL reaction system was added with the corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of a PCR product and a fluorescent probe (HEX-N12 -BHQ1) (0.5 μM). The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0183] [0183] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 14, only when the crRNA corresponded to the corresponding target sequence, would there be a higher fluorescence response value and, if there was a single point mutation, its response value would be greatly reduced. The corresponding SNP genotype can be determined by the fluorescence value, and these results were confirmed by the sequencing results. Example 8 Testing for a cancer-related gene
[0184] [0184] A TP53 gene was selected as the test gene. The TP53 gene has a nonsense mutation in a human T24 cell, which leads to inactivation of the gene. A cell with a normal gene at this site (HEK293T), an individual gene and a T24 mutant cell were tested, respectively.
[0185] [0185] 1. Preparation of crRNA: first, a transcription model was prepared by annealing T7-crRNA-F to the synthesized T7-crRNA-34-TP53-T24-C-16nt and T7-crRNA-34-TP53- oligonucleotides T24-G-16nt (Table 5).
[0186] [0186] 2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of each primer 34-TP53-T24-F and 34-TP53-T24-R (table 4), 1 ng of a human genome (HEK293T, T24) or directly shaved oral epithelial mucosa as a model, and a high-fidelity KOD FX enzyme (ToYoBo) was used for the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. After PCR completion, the product was used directly for a Cas12a reaction.
[0187] [0187] 3. Cas12a reaction: a 20 μL reaction system was added with the corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of a PCR product and a fluorescent probe (HEX-N12 -BHQ1) (0.5 μM). The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0188] [0188] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nM and an emission light at 556 nM). As shown in FIGURE 15, when the TP53 gene that was normal at that site was the model, the detected value of crRNA-C was significantly higher than that of crRNA-G, while the crRNA-G of the mutant T24 cell was significantly increased. Example 9 Human SNP test (genes related to gout)
[0189] [0189] The SNP test selected 5 human SNP sites that were related to risk of gout, such as rs1014290, rs6449213, rs737267, rs1260326 and rs642803, to test the HOLMES method.
[0190] [0190] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). The RNA was purified using RNA Clean & ConcentratorTM-5 (Zymo Research), quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C.
[0191] [0191] 2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of each primer (Table 4), 1 ng of a human genome (HEK293T) or directly shaved oral epithelial mucosa were used was used as a model, and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s. After PCR completion, the product was used directly for a Cas12a reaction. (Primers 1-rs5082-F-T, 2-rs1467558-F-T and 3-rs2952768-R-C were introduced directly into the corresponding SNP mutation products)
[0192] [0192] 3. Cas12a reaction: a 20 μL reaction system was added with the corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of a PCR product and a fluorescence probe (HEX- N12-BHQ1) (0.5 μM). The reaction was carried out at 37 ° C for 15 min and then ended at 98 ° C for 2 minutes.
[0193] [0193] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 16, only when the crRNA corresponded to the corresponding target sequence would there be a higher fluorescence response value and, if there was a single point mutation, its response value would be greatly reduced. The corresponding SNP genotype can be determined by the fluorescence value, and these results were confirmed by the sequencing results. Example 10 SNP test of clinical samples from volunteers (a gout-related gene) using a kit
[0194] [0194] A premixed solution was added to a 96-well plate to prepare a kit, and then genomic DNAs from 21 volunteers were added to the kit to test the rs1014290 site, which was related to gout risk.
[0195] [0195] 1. Preparation of a kit: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). The RNA was purified using RNA Clean & ConcentratorTM-5 (Zymo Research), quantified with NanoDrop 2000C and diluted to a concentration of 10 μM.
[0196] [0196] 2. Premix in a 96-well PCR plate: a 19 μL system was added with the necessary reagents for a PCR reaction, the primers being 41-rs1014290-F and 41-rs1014290-R.
[0197] [0197] 3. Pre-mixing in a 96-well plate for fluorescence detection: a crRNA (1 μM), LbCas12a (0.5 μm) and a fluorescence probe (HEX-N12-) were added to the 19 μL system BHQ1) (0.5 μM) and it was added to the 96-well plate.
[0198] [0198] 4. PCR amplification: the volunteers' genomic DNAs were added to the 96-well pre-mixed PCR plate and then it was subjected to the PCR reaction. The PCR reaction procedure took place at 95 ° C for 2 minutes and then started 35 cycles at 98 ° C for 10 s, 60 ° C for 15 s and 68 ° C for 10 s.
[0199] [0199] 5. Cas12a reaction: 1 μL of a PCR reaction solution was taken and added to the pre-mixed 96-well plate for fluorescence detection, reacted at 37 ° C for 15 minutes and then the reaction was closed at 98 ° C for 2 minutes.
[0200] [0200] 6. Fluorescence detection: it was detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 17, as the population with genotype A: A was at greater risk of having gout, other people than volunteers # n. 5, 7 and 9 are genotype A: G or G: G; therefore, more attention should be paid to the risk of gout. Example 11 Detection of Escherichia coli and similar microorganisms in ambient water by LAMP combined with a Cas protein
[0201] [0201] The Escherichia coli gyrB gene was selected as a detection target to indirectly test whether there were concentrations of Escherichia coli and similar microorganisms in the water.
[0202] [0202] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7-crRNA-gyrB oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1 X Taq DNA polymerase reaction buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermal cycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, finally diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C for later use.
[0203] [0203] 2. LAMP amplification: sterile water and a contaminated liquid containing Escherichia coli were used as a negative control and as a sample to be detected, respectively. The total volume of each reaction system was 25 μL, primers of 1.6 μM of LAMP-FIP and LAMP-BIP each, 0.2 μM of LAMP-F3 and LAMP-B3 each, 0.4 μM of each were used. LAMP-LoopF and LAMP-LoopB each and the kit used for the LAMP reaction was the WarmStart® LAMP Kit (NEB). The LAMP reaction procedure was at 65 ° C for 30 minutes. After the completion of LAMP, the annealing was carried out at 80 ° C for 10 minutes and then the product was used directly for a reaction of Cas12a.
[0204] [0204] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, Cas12a (0.25 μM), 1 μL of a LAMP product, a probe fluorescence (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 minutes.
[0205] [0205] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). The results were as shown in FIGURE 19. Example 12 Detecting SNP using LAMP amplification combined with a Cas protein
[0206] [0206] 1. Preparation of the crRNA: first, a transcription model was prepared by annealing the T7-crRNA-F to a synthesized T7-crRNA-rs5082-T / T7-crRNA-rs5082-G / T7-crRNA-rs1467558 oligonucleotide -T / T7-crRNA-rs14 67558-C (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1 X Taq DNA polymerase reaction buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermal cycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, finally diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C for later use.
[0207] [0207] 2. LAMP amplification: a HEK293T human genome was used as a sample. The total volume of each reaction system was 25 μL, primers of 1.6 μM of LAMP-FIP and LAMP-BIP each, 0.2 μM of LAMP-F3 and LAMP-B3 each, 0.4 μM of each were used. LAMP-LoopF and LAMP-LoopB each and the WarmStart® LAMP Kit (NEB) was used for LAMP reaction. The LAMP reaction procedure was at 65 ° C for 30 minutes. After the completion of LAMP, the annealing was carried out at 80 ° C for 10 minutes and then the product was used directly for a reaction of Cas12a.
[0208] [0208] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, Cas12a (0.25 μM), 1 μL of a LAMP product, a probe fluorescence (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 minutes.
[0209] [0209] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). The results were shown in FIGURE 20. Example 13 Detection of Escherichia coli and similar microorganisms in ambient water by amplification of RPA combined with a Cas protein
[0210] [0210] The Escherichia coli gyrB gene was selected as a detection target to indirectly test whether there were concentrations of Escherichia coli and similar microorganisms in the water.
[0211] [0211] 1. crRNA preparation: first, a transcription model was prepared by annealing T7-crRNA-F to a synthesized T7-crRNA-gyrB oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 50 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler. The crRNA was synthesized using a high-performance T7 transcription kit, and the reaction was carried out overnight at 37 ° C (for about 16 h). Then, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop2000C, finally diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C for later use.
[0212] [0212] 2. Amplification of RPA: sterile water and a contaminated liquid containing Escherichia coli were used as a negative control and as a sample to be detected, respectively. The total volume of each reaction system was 25 μL, 0.5 μM of each RPA-gyrB-F (or RPA-gyrB-F2) primer and RPA-gyrB-R2 was used and a TwistAmp® Basic kit (TwistDX) was used for the RPA reaction. The RPA reaction procedure took place at 37 ° C for 30 minutes. After the completion of RPA, the annealing was carried out at 80 ° C for 10 minutes and then the product was used directly for a reaction of Cas12a.
[0213] [0213] 3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified in step 1, Cas12a (0.25 μM), 1 μL of a RPA product, a probe fluorescence (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB 3.1 buffer and 0.5 μL of an RNase inhibitor. The reaction was carried out at 37 ° C for 15 minutes.
[0214] [0214] 4. Fluorescence detection: 20 μL of the inactivated reaction solution was added to a 96-well plate and then detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). The results were shown in FIGURE 21. Example 14: Cas12b having collateral cleavage activity
[0215] [0215] 1. Preparation of a guide RNA (sgRNA)
[0216] [0216] First, an RNA-T1 guide plasmid pUC18 was constructed using pUC18 as the plasmid backbone. In the plasmid, a T7 promoter and a model DNA sequence for the transcription of the guide RNA were inserted into pUC18 (Note: the guide RNA transcribed from this model was directed to a sequence called T1 in this study). The method first performed a round of PCR using plasmid pUC18 as a template and PUC18-1-F and pUC18-1- R as primers; linking PCR products to a T4 DNA ligase, transforming the product into DH10b and sequencing to obtain the correct clone, which was called guide pUC18 pre-RNA-T1-pre. Then, a second round of PCR was performed using pUC18 guide RNA-T1-pre as a model and pUC18-2-F and pUC18-2- R as primers, linking and transforming the PCR products in the same way, to finally obtain the plasmid pUC18 guide RNA-T1 that was correct as sequenced.
[0217] [0217] Then, using the pUC18 guide plasmid RNA-T1 as a template, a guide RNA was synthesized using a high-performance T7 transcription kit (Thermo), and the reaction was carried out overnight at 37 ° C (for 12 -16 hours).
[0218] [0218] Finally, a DNase I (2 μL of DNase I per 50 μL of the transcription system was added) was added to the transcription system, the system was placed in a water bath at 37 ° C for 30 minutes to eliminate a Plasmid DNA and RNA were purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM and stored in a refrigerator at -80 ° C for later use.
[0219] [0219] 2. Preparation of a target DNA
[0220] [0220] (1) If the target DNA is single-stranded, a 66 bp long oligonucleotide is directly synthesized as the target DNA (target T1-R), which contained the 20 bp target sequence (T1) recognized by the RNA guide.
[0221] [0221] (2) If the target DNA is double-stranded, two complementary 66 bp oligonucleotides (target T1-F; target T1-R) are directly synthesized, containing the identified 20 bp target sequence (T1) by the guide RNA. The two oligonucleotides were annealed to obtain a short target DNA. Specifically, the paired oligonucleotide (1 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 20 μL and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes and , then cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermocycler.
[0222] [0222] 3. Cas12b reaction
[0223] [0223] (1) annealing of the guide RNA: the guide RNA was diluted to an appropriate concentration (10 μM) and annealed on the PCR instrument. Annealing procedure: denaturation at 75 ° C for 5 minutes and then cooling from 75 ° C to 20 ° C at a decreasing rate of 1 ° C per minute.
[0224] [0224] (2) incubation of the guide RNA with C2c1: the annealed guide RNA was mixed with C2c1 in an equal molar concentration and allowed to stand at 30 ° C for 20-30 minutes.
[0225] [0225] (3) Cas12b reaction: a 20 μL reaction system was added with a mixture of the guide RNA and C2c1 incubated in step (2) (both final concentrations were 250 μM or 500 μM), a target DNA (with 50 nM final concentration), a FAM-labeled oligonucleotide (target DNMT1-3- R-FAM-5 ') or a suppression fluorescence probe (HEX-N12-BHQ1 with 500 nM final concentration), 2 μL of buffer 10 X NEB 3.1 and 0.5 μL of an RNase inhibitor (40 U / μL). After mixing homogeneously, the reaction was carried out at 48 ° C for 30 minutes. After that, it was inactivated by heating to 98 ° C for 5 minutes in a PCR instrument.
[0226] [0226] 4. Detection of Cas12b transcleavage activity by denatured urea gel electrophoresis: 20 μL of the inactivated reaction solution was separated by a denatured urea gel electrophoresis method and then transformed into an image by a system fluorescence imaging system ImageQuant LAS 4000 mini (GE Healthcare). The results were those shown in FIGURE 22.
[0227] [0227] 5. Detection of Cas12b transcleavage activity by a fluorescence microplate reader method: 20 μL of the inactivated reaction solution was added to a 96-well plate and detected by a microplate reader (with an excitation light at 535 nm and a light emission at 556 nm). The results were shown in FIGURE 23. Example 15: Sensitivity test of a Cas12b reaction (transcleavage)
[0228] [0228] When detecting the excited fluorescence intensity of the fluorescence probe (HEX-N12-BHQ1), the target DNA concentration required for Cas12b to exert the transcleavage activity, that is, the sensitivity of a Cas12b transcleavage reaction, was determined .
[0229] [0229] 1. Preparation of a guide RNA
[0230] [0230] First, using the pUC18 guide RNA-T1 as a template and the guide RNA DNMT1-3-F and the guide RNA DNMT1-3-R as primers, 20 bases of the guide RNA that targets T1 target DNA were replaced by a guide RNA directed to DNMT1-3 by PCR, in order to obtain another pUC18 guide plasmid RNA-DNMT1-3.
[0231] [0231] Then, using the pUC18 guide plasmid RNA-DNMT1-3 as a template, a guide RNA was synthesized using a high-performance T7 transcription kit (Thermo), and the reaction was carried out overnight at 37 ° C ( for 12-16 hours).
[0232] [0232] Finally, a DNase I (2 μL of DNase I per 50 μL of the transcription system was added) was added to the transcription system, the system was placed in a water bath at 37 ° C for 30 minutes to eliminate a Plasmid DNA and RNA were purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C and stored in a refrigerator at -80 ° C for later use.
[0233] [0233] 2. Preparation of a target DNA
[0234] [0234] For target DNA, first target DNA was added directly to the Cas12b reaction system without amplification. The method was as follows:
[0235] [0235] (1) If the target DNA is single-stranded, a 50 bp long oligonucleotide is directly synthesized as the target DNA (DNMT1-3 (TTC PAM) -
[0236] [0236] (2) If the target DNA is double-stranded, two complementary oligonucleotides of 50 bp in length (DNMT1-3 (TTC PAM) -F; DNMT1-3 (TTC PAM) -R) are directly synthesized, containing the target sequence of 20 bp (DNMT1-3) recognized by the guide RNA. The two oligonucleotides were annealed to obtain a short target DNA. Specifically, the paired oligonucleotide (2 μM) was annealed in a 1X PCR buffer (Transgen Biotech) to a total volume of 20 μL and then subjected to an annealing procedure: denaturation at 95 ° C for 5 minutes and, then, cooling from 95 ° C to 20 ° C at a rate of 1 ° C per minute using a thermal cycler.
[0237] [0237] (3) Single- or double-stranded target DNA was gradually diluted to 2 μM, 0.2 μM, 0.02 μM, 0.002 μM and 0.0002 μM for later use.
[0238] [0238] The second step was to insert a fragment containing the target sequence (DNMT1-3) into a plasmid vector for amplification by the LAMP reaction.
[0239] [0239] (1) The fragment containing the target sequence (DNMT1-3) was inserted into a pEasy-Blunt zero cloning vector using Transgen's pEasy-Blunt zero cloning kit in order to obtain the correct clone after verification by sequencing.
[0240] [0240] (2) A LAMP expansion reaction
[0241] [0241] Using the above plasmid as a model, the LAMP amplification reaction was performed. The models were added at 0 nM, 1 nM and 0.1 nM respectively and diluted with a 10-fold gradient at 10-11 nM. The total volume of each reaction system was 25 μL, primers of 1.6 μM of LAMP-DNM-FIP and LAMP-DNM-BIP were used, 0.2 μM of LAMP-DNM-F3 and LAMP-DNM- B3 each, 0.4 μM of LAMP-DNM-LoopF and LAMP-DNM-LoopB each and the kit used for the LAMP reaction was the WarmStart® LAMP Kit (NEB). The LAMP reaction procedure was at 65 ° C for 30 minutes. After the completion of LAMP, inactivation was carried out at 80 ° C for 10 minutes and then the product was used directly for a Cas12b reaction.
[0242] [0242] 3. Cas12b reaction
[0243] [0243] (1) Annealing of the guide RNA: the guide RNA was diluted to an appropriate concentration (5 μM) and annealed on the PCR instrument. The annealing procedure: denaturation at 75 ° C for 5 minutes and then cooling from 75 ° C to 20 ° C at a decreasing rate of 1 ° C per minute.
[0244] [0244] (2) incubation of the guide RNA with Cas12b: the annealed guide RNA was mixed with Cas12b in an equal molar concentration and allowed to stand at 30 ° C for 20-30 minutes.
[0245] [0245] (3) Cas12b reaction: a 20 μL reaction system was added with the mixture of the guide RNA and Cas12b incubated in step (2) (both the final concentrations of the guide RNA and Cas12b were 250 μM), 1 μL of a target DNA or 1 μL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1) (with a final concentration of 500 nM), 2 μL of a NEB 10X 3.1 buffer and 0.5 μL of a RNase inhibitor (40 U / μL). After mixing homogeneously, the reaction was carried out at 48 ° C for 30 minutes. After that, it was inactivated by heating over a fire at 98 ° C for 5 minutes in a PCR instrument.
[0246] [0246] 4. Detection of Cas12b transcleavage activity by a fluorescence microplate reader method:
[0247] [0247] 20 μL of the inactivated reaction solution was added to a 96-well plate and detected by a microplate reader (with an excitation light at 535 nm and an emission light at 556 nm). Combined with LAMP amplification, Cas12b could produce significant collateral single-stranded DNA transcleavage activity for a target DNA concentration as low as 10 aM. The results were as shown in FIGURE 24. Cas12a cis cleavage characteristics in the cleavage of a single stranded target DNA:
[0248] [0248] First, to test the cleavage characteristics of Cas12a single-stranded DNA, several crRNAs targeting a short single-stranded DNA (DNMT1-3) (Table 1) are designed, marked with 5 (6) -carboxyflorescein ( FAM) at terminal 3 '. After cleavage by FnCas12a, the reaction product is analyzed by denatured urea-polyacrylamide gel electrophoresis (urea-PAGE). It appears that Cas12a single-stranded DNA cleavage is programmed. That is, the cleavage site is close to the 22nd base (21st to 23rd bases) of the target sequence counting from the base of the 3 'end to the 5' end of the first target sequence paired with the crRNA guide sequence, as shown in FIGURES 1A and 1C. Cleavage of a double-stranded DNA by Cas12a requires a PAM sequence, while cleavage of a single-stranded DNA by Cas12a
[0249] [0249] When target single-stranded DNA is labeled at the 3 'terminal, Cas12a cleaves near the 22nd base, as shown in FIGURE 1. However, when it is labeled at the 5' terminal, no predicted size cleavage product strand is observed, but a short product (<6 nt) labeled with FAM is produced, as shown in FIGURE 3B. Through detailed experiments, once the ternary Cas12a / crRNA / single stranded DNA complex is formed, the single stranded DNA strand (DNMT1-3) (Table 1) marked at the 5 'end is cleaved and a product marked with FAM is produced, as shown in FIGURE 3C. In addition, the ternary complex also cleaves a single-stranded DNA that has no sequence that is complementary to the crRNA (that is, a single-stranded collateral DNA) in any other reaction system, as shown in FIGURES 3C and 3D. This cleavage phenomenon is called transcleavage, which is different from programmable cis cleavage. When target single-stranded DNA is labeled at the 3 'terminal, transcleavage is also observed, but many cis cleavage products remain, as shown in FIGURE 3B. This may be due to the complex formed by Cas12a / crRNA / single stranded DNA and the single stranded DNA is protected, so that its labeled 3 'terminal is protected from exposure to an active nuclease site of the ternary complex. These cleavage processes can be like those shown in FIGURE 3A.
[0250] [0250] In addition to the FnCas12a tested above, 9 Cas12as from other species sources are also tested (Table 2 and FIGURE 4A). Except for Lb4Cas12a, all Cas12as have good endonuclease activity in plasmid DNA (as shown in FIGURE 4B), and all Cas12a ternary complexes exhibit cis and trans cleavage activities in the single strand (as shown in FIGURES 4C and 4D). This shows that the cis and trans activity of Cas12a in single stranded DNA is a common phenomenon. The key cis and trans sites and mechanism for cleaving a single stranded DNA by Cas12a.
[0251] [0251] To determine the key amino acid residues related to cis and trans activities in the single-stranded DNA in Cas12a, several candidate residues of Cas12a are mutated to perform the activity test. First, three FnCas12a single amino acid mutants (H843A, K852A and K869A) are purified and tested, and their residues are related to an RNase activity. The results of the study of trans activity in single stranded DNA show that no obvious difference is found in cis and trans cleavage activities in single stranded DNA between wild-type FnCas12a and the three mutants, as shown in FIGURES 5A and 5C .
[0252] [0252] Then, when the active endonuclease sites in FnCas12a, that is, the sites of the RuvC domain (D917A, E1006A or D1255A) and the Nuc domain (R1218A) are mutated, the two cis and trans cleavage activities of this Cas12a mutated in single stranded DNA are affected, as shown in FIGURES 5B and 5D. These results indicate that the Cas12a key site for cleavage of a double stranded target DNA is closely related to cis and trans cleavage activities in single stranded DNA.
[0253] [0253] Recent structural studies of Cas12b (i.e., C2c1) (including complexes with an extended target DNA or an extended non-target DNA) show that both strands are located in a RuvC well, as shown in FIGURES 6A and 6B. When comparing the endonuclease catalytic residues of Cas12b (ie C2c1) and Cas12a, these sites are more likely to play similar roles in the cleavage and functions of Cas12b (ie C2c1) and Cas12a. The results of a single amino acid mutation experiment in vitro show that it is consistent with the above hypothesis. That is, it is likely that Cas12a will cleave two strands through only one RuvC catalytic well.
[0254] [0254] The transcleavage activity of a Cas12a complex: in the structure of a Cas12b complex (ie C2c1) with additional single-stranded DNA, a sequence-independent single-stranded DNA is also located on the surface of a catalytic cavity , as shown in FIGURE 6C, which is similar to that of a single stranded DNA substrate in Cas12a. Combined with a single amino acid mutation experiment, it is proposed that the target DNA, non-target DNA and single-stranded collateral DNA are all cleaved in the single RuvC well in Cas12a, as shown in FIGURES 6D, 6E and 6F. The Cas12a ternary complex has a single-stranded DNA strand cleavage activity, while the reason why a monomer or binary complex does not have the single-stranded DNA strand cleavage activity can be explained by comparing the structures of the monomer, binary complexes and ternary. The structure of a Cas12a monomer is disordered, the binary Cas12a / crRNA complex has a triangular structure, as shown in FIGURE 6G, while the ternary Cas12a / crRNA / DNA complex is converted into a double leaf structure, thus exposing the catalytic cavity to perform the transcleavage of single-stranded collateral DNA (as shown in FIGURE 6H). Establishment of a nucleic acid detection method
[0255] [0255] Based on the characteristics of Cas12a, a method for specific detection of nucleic acid molecules, called
[0256] [0256] Throughout the reaction system, the method can be divided into two main steps, one is the amplification of a model nucleic acid and the other is the specific detection of nucleic acid by a Cas12a protein. Here, a PCR method is used for nucleic acid amplification, but in fact, any amplification method can be combined with the detection of nucleic acid in the second step, such as the RPA isothermal amplification method, etc. The initial nucleic acid is not limited to double-stranded DNA, but it can also be single-stranded DNA; or even an RNA can still be detected after reverse transcription, so this method is suitable for several types of nucleic acid molecules. For the nucleic acid detection phase, three components are the keys of the experiment, namely, Cas12a, crRNA and a nucleic acid probe. In addition to the 10 Cas12a mentioned in the example (these 10 proteins are selected at random), other Cas12a proteins are also suitable for this method. In addition, other types of Cas proteins (for example, a C2c1 protein) are also suitable for the claimed scope of the present invention: as shown by the experimental results, Alicyclobacillus acidoterrestris Cas12b (i.e., C2c1) also has DNA transcleavage activity collateral single strand similar to Cas12a and its crRNA / target DNA complex can also cleave collateral single strand DNA.
[0257] [0257] For the crRNA that serves as a guide, it will be more stable in the system after being designed, for example, being modified manually. With regard to the selection of nucleic acid probes, the present invention selects a short single-stranded DNA labeled with HEX and BHQ1, and any other detectable labeling method is theoretically applicable, as long as the nucleic acid probe is cleaved to produce detectable differences. Alternatively, the nucleic acid probe can also be designed to become fluorescent after binding to a compound, in order to detect whether the probe is cleaved.
[0258] [0258] Furthermore, it should be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the present order.
[0259] [0259] Primers used for detection by DNA amplification using LAMP combined with Cas12a: Table 6 Primers used to amplify gyrB-1
[0260] [0260] All documents mentioned in the present invention are incorporated into this instrument by reference as if each document were incorporated individually by reference.
Furthermore, it should be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

权利要求:
Claims (21)
[1]
1. Method for detecting a target nucleic acid molecule, characterized by the fact that it comprises the addition of a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution in a system containing the target nucleic acid molecule to be detected and then detection of the nucleic acid probe.
[2]
2. Method for detecting a target nucleic acid molecule according to claim 1, characterized by the fact that the Cas protein is Cas12a or a Cas protein that has a collateral single-stranded DNA cleavage activity similar to that of Cas12a; Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; and Cas12a is preferably LbCas12a.
[3]
Method for detecting a target nucleic acid molecule according to claim 1, characterized in that the guide RNA refers to an RNA that guides the Cas protein to specifically bind to a target DNA.
[4]
4. Method for detecting a target nucleic acid molecule according to claim 1, characterized in that the nucleic acid probe is single-stranded DNA; single-stranded DNA is preferably fluorescently labeled single-stranded DNA; the single-stranded DNA is preferably a fluorescent probe that is labeled with a fluorescent HEX group at a 5 'terminus and labeled with a BHQ1 suppressor group at a 3' terminus; preferably, the method for detecting the nucleic acid probe is preferably a fluorescence detection method; and the fluorescence detection method is preferably a detection method that uses a microplate reader or a fluorescence spectrophotometer.
[5]
Method for detecting a target nucleic acid molecule according to any one of claims 1 to 4, characterized in that the target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained by amplification.
[6]
6. Method for detecting a target nucleic acid molecule according to claim 5, characterized in that the detection method can detect a pathogenic microorganism, genetic mutation or a specific target DNA.
[7]
7. Method according to claim 1, characterized by the fact that the Cas protein comprises Cas12b (i.e., C2c1).
[8]
8. Use of a Cas protein in a method characterized by the fact that it is to detect a target nucleic acid molecule.
[9]
9. Use, according to claim 8, characterized by the fact that when a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system; and, preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind to the target DNA.
[10]
10. Kit to detect a target nucleic acid molecule, characterized by the fact that it comprises a guide RNA, a Cas protein and a nucleic acid probe.
[11]
11. Detection system to detect a target nucleic acid molecule, characterized by the fact that it comprises: (a) a Cas protein, which is Cas12a or a Cas protein with a single-stranded DNA cleavage activity similar to that of Cas12a ; (b) a guide RNA that guides the Cas protein to specifically bind to the target nucleic acid molecule; and (c) a nucleic acid probe that is single-stranded DNA; wherein the target nucleic acid molecule is target DNA.
[12]
12. Detection system according to claim 11, characterized by the fact that the Cas protein that has a collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (ie, C2c1 ).
[13]
13. Detection system according to claim 11, characterized in that the nucleic acid probe comprises a single-stranded DNA that carries a detectable marker.
[14]
14. Kit to detect a target nucleic acid molecule, characterized by the fact that it comprises:
i) a first container and a Cas protein in the first container, the Cas protein being Cas12a or Cas protein having a collateral single-stranded DNA cleavage activity similar to that of Cas12a; ii) an optional second container and a guide RNA in the second container, the guide RNA guiding the Cas protein to specifically bind to the target nucleic acid molecule; iii) a third container and a nucleic acid probe in the third container; iv) an optional fourth container and a buffer solution in the fourth container; wherein the target nucleic acid molecule is target DNA.
[15]
15. Method for detecting whether a target nucleic acid molecule exists in a sample, characterized by the fact that it comprises the following steps: (a) providing the detection system to detect a target nucleic acid molecule, according to claim 11 , wherein the detection system further comprises a sample to be detected; and (b) detecting whether the nucleic acid probe in the detection system is cleaved by a Cas protein, wherein the cleavage is a transcleavage of a single-stranded collateral DNA; wherein if the nucleic acid probe is cleaved by the Cas protein, then this indicates the presence of the target nucleic acid molecule in the sample; and if the nucleic acid probe is not cleaved by the Cas protein, this indicates the absence of the target nucleic acid molecule in the sample.
[16]
16. Method according to claim 15, characterized in that a method for amplifying nucleic acid is selected from a group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification , NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
[17]
17. Method according to claim 15, characterized by the fact that when the upstream and downstream regions (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, and most preferably in a range of -10 nt to +10 nt) from a target site do not have a PAM sequence,
nucleic acid amplification is performed using a PAM introducer primer.
[18]
18. Method, according to claim 17, characterized by the fact that the PAM introducer primer has a structure of formula I in 5'-3 ': P1-P2-P3 (I) in which, P1 is a sequence of segment 5 'which is located at terminal 5' and is complementary or not complementary to the sequence of the target nucleic acid molecule; P2 is a PAM sequence; and P3 is a 3 'segment sequence that is located at the 3' terminal and is complementary to the sequence of the target nucleic acid molecule.
[19]
19. Method, according to claim 15, characterized by the fact that, when the upstream and downstream regions (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, and more preferably in the range of -10 nt to +10 nt) the target site contains the PAM sequence, then a primer containing or not the PAM sequence can be used and the amplified amplification product will contain the PAM sequence.
[20]
20. Use of a Cas protein in the preparation of a detection reagent or kit to detect a target nucleic acid molecule based on the cleavage of single-stranded DNA, characterized by the fact that the Cas protein is Cas12a or a protein Cas having a collateral single-stranded DNA cleavage activity similar to that of Cas12a.
[21]
21. Use according to claim 20, characterized by the fact that the Cas protein that has a collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (or C2c1).

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112020000809A2|2020-09-08|use of cas protein, method for detecting nucleic acid molecules, and kit

---

Kiddle et al.2012|GMO detection using a bioluminescent real time reporter | of loop mediated isothermal amplification | suitable for field use

---

JP2020530779A5|2021-05-13|

---

KR20110106922A|2011-09-29|Single-cell nucleic acid analysis

---

JP6335918B2|2018-05-30|Target enrichment without restriction enzymes

---

JP5945271B2|2016-07-05|Helicase-dependent isothermal amplification using nicking enzymes

---

CN107208137A|2017-09-26|Detect the composition and method of RNA virus

---

BRPI0814527B1|2018-07-17|methods for amplifying a double-stranded and single-stranded nucleic acid target sequence as well as kit for amplifying a target nucleic acid sequence

---

KR20120084320A|2012-07-27|Thd primer target detection

---

WO2020056924A1|2020-03-26|Method for detecting nucleic acid

---

JP2017136086A|2017-08-10|Method and kit for detecting target nucleic acids

---

ES2776427T3|2020-07-30|Strand Invasion Based DNA Amplification Method

---

JP2008161165A|2008-07-17|Method for detecting gene using competing oligonucleotide

---

JP2007319096A|2007-12-13|Nucleic acid amplification method utilizing nicking activity of endonuclease

---

US20210102237A1|2021-04-08|Means and methods for nucleic acid target detection

---

US20210230677A1|2021-07-29|Application of cas protein, method for detecting target nucleic acid molecule and kit

---

JP2008161164A|2008-07-17|Method for detecting gene with primer containing artificial mismatched nucleic acid

---

US9074248B1|2015-07-07|Primers for helicase dependent amplification and their methods of use

---

US20210285039A1|2021-09-16|Looped primer and loop-de-loop method for detecting target nucleic acid

---

CN113337638A|2021-09-03|Method and kit for detecting novel coronavirus |

---

JP7004570B2|2022-02-10|Substrate molecule

---

Fu et al.2021|Ultra-specific nucleic acid testing by target-activated nucleases

---

US9212398B2|2015-12-15|Cross priming amplification of target nucleic acids

---

JP2001258569A|2001-09-25|Oligonucleotide for detecting vibrio parahaemolyticus

---

CN114174535A|2022-03-11|CRISPR multi-target detection method and kit thereof

同族专利:

---

公开号 | 公开日

---

EP3653722A1|2020-05-20|

---

CA3069788A1|2019-01-17|

---

PH12020500103A1|2020-10-19|

---

CN107488710B|2020-09-22|

---

CN111094588A|2020-05-01|

---

MA49579A|2020-05-20|

---

ZA202000682B|2021-01-27|

---

EA202090290A1|2020-07-14|

---

KR20200035956A|2020-04-06|

---

SG11202000336RA|2020-02-27|

---

CN112501254A|2021-03-16|

---

WO2019011022A1|2019-01-17|

---

AU2018299445A1|2020-02-06|

---

IL272005D0|2020-02-27|

---

EP3653722A4|2021-04-21|

---

CN107488710A|2017-12-19|

---

JP2020530779A|2020-10-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

CN110066775A|2012-10-23|2019-07-30|基因工具股份有限公司|Composition and application thereof for cutting target DNA|

---

WO2016123243A1|2015-01-28|2016-08-04|The Regents Of The University Of California|Methods and compositions for labeling a single-stranded target nucleic acid|

---

JP2018531596A|2015-09-24|2018-11-01|シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニーＳｉｇｍａ−Ａｌｄｒｉｃｈ Ｃｏ．， ＬＬＣ|Methods and reagents for intermolecular proximity detection using RNA-guided nucleic acid binding proteins|

---

WO2017070605A1|2015-10-22|2017-04-27|The Broad Institute Inc.|Type vi-b crispr enzymes and systems|

---

CN112501254A|2017-07-14|2021-03-16|上海吐露港生物科技有限公司|Application of Cas protein, and detection method and kit of target nucleic acid molecule|

---

US10253365B1|2017-11-22|2019-04-09|The Regents Of The University Of California|Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs|CN112501254A|2017-07-14|2021-03-16|上海吐露港生物科技有限公司|Application of Cas protein, and detection method and kit of target nucleic acid molecule|

---

US10253365B1|2017-11-22|2019-04-09|The Regents Of The University Of California|Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs|

---

CN108220400A|2018-01-02|2018-06-29|武汉启丰生物技术有限公司|A kind of quick detection of nucleic acids sizing technique based on CRISPR-Cas10 nucleic acid-protein compounds|

---

CN108796036B|2018-04-03|2021-11-19|交弘生物科技有限公司|Nucleic acid detection method based on prokaryotic Argonaute protein and application thereof|

---

CN108588182A|2018-04-13|2018-09-28|中国科学院深圳先进技术研究院|Isothermal duplication and detection technique based on the substitution of CRISPR- chains|

---

CN110551800A|2018-06-03|2019-12-10|上海吐露港生物科技有限公司|Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule|

---

WO2020030984A2|2018-08-09|2020-02-13|G+Flas Life Sciences|Compositions and methods for genome engineering with cas12a proteins|

---

CN109055499B|2018-08-30|2021-01-19|杭州杰毅生物技术有限公司|Isothermal nucleic acid detection method and kit based on CRISPR-Cas|

---

CN109837328B|2018-09-20|2021-07-27|中国科学院动物研究所|Nucleic acid detection method|

---

CN111117984A|2018-11-01|2020-05-08|浙江大学|CRISPR system, method and device for visually and specifically detecting nucleic acid target|

---

CN109666662A|2018-12-12|2019-04-23|广州普世利华科技有限公司|Application of the novel ScCas12a in terms of detection of nucleic acids|

---

EP3931313A2|2019-01-04|2022-01-05|Mammoth Biosciences, Inc.|Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection|

---

CN109628564B|2019-02-25|2022-02-01|北京市理化分析测试中心|Primer set for detecting SNP polymorphism and method for detecting SNP polymorphism by using primer set|

---

CN109929949B|2019-03-14|2019-12-31|广州医科大学附属肿瘤医院|EBV detection method based on CRISPR-Cas12a and G quadruplex-heme and application thereof|

---

CN110117818A|2019-04-16|2019-08-13|天津大学|Detect the CRISPR high flux biochip of single gene mutation|

---

CN110157821B|2019-05-06|2020-08-28|浙江大学|Kit for detecting human staphylococcus|

---

CN110317890A|2019-06-24|2019-10-11|浙江大学|A kind of kit for enterococcus faecalis detection|

---

CN110241238B|2019-06-24|2020-03-03|浙江大学|Kit for detecting streptococcus pyogenes|

---

CN110331219A|2019-06-24|2019-10-15|浙江大学|A kind of kit for pseudomonas aeruginosa detection|

---

CN110358848A|2019-06-24|2019-10-22|浙江大学|A kind of kit for enterococcus faecium detection|

---

CN112301101A|2019-07-24|2021-02-02|上海吐露港生物科技有限公司|CRISPR multi-target detection method and kit thereof|

---

CN110894557A|2019-09-20|2020-03-20|武汉大学|CRRNAfor detecting African swine fever virus based on CRISPRmode and kit|

---

CN110551812B|2019-09-26|2020-08-18|暨南大学|CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof|

---

CN112986551A|2019-12-11|2021-06-18|清华大学|Method and kit for detecting concentration of target molecules in mixed system|

---

CN110894530B|2019-12-25|2020-12-08|武汉博杰生物医学科技有限公司|Detection kit and detection method for gene mutation of colon cancer-related molecular marker|

---

WO2021154866A1|2020-01-27|2021-08-05|Sherlock Biosciences|Improved detection assays|

---

WO2021155775A1|2020-02-03|2021-08-12|苏州克睿基因生物科技有限公司|Method and kit for dectecting target nucleic acid|

---

CN111440793B|2020-03-20|2021-11-05|武汉博杰生物医学科技有限公司|Novel coronavirus nucleic acid detection kit|

---

WO2021252836A1|2020-06-12|2021-12-16|Sherlock Biosciences|Crispr-based sars-cov-2 detection|

---

CN112795624B|2020-07-17|2021-11-19|山东舜丰生物科技有限公司|Method for detecting target nucleic acid using nucleic acid detector containing abasic spacer|

---

CN111909987A|2020-07-31|2020-11-10|浙江科途医学科技有限公司|Reagent composition, kit and detection system for KIF5B-RET fusion gene detection|

---

CN112251494A|2020-10-09|2021-01-22|湖南大学|Nucleic acid detection method based on CRISPR-Cas autocatalytic amplification network and application thereof|

---

CN113046477A|2021-02-26|2021-06-29|广州医科大学附属第一医院（广州呼吸中心）|Specific crRNA for identifying D614G mutation site, system, kit and application|

---

CN112852980B|2021-04-23|2021-11-26|上海海关动植物与食品检验检疫技术中心|Detection method for detecting nucleic acid of Atlantic salmon based on CRISPR fluorescence method and detection kit thereof|

法律状态:
2021-11-23| 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

---

CN201710573752.0|2017-07-14|

---

CN201710573752.0A|CN107488710B|2017-07-14|2017-07-14|Application of Cas protein, and detection method and kit of target nucleic acid molecule|

---

PCT/CN2018/082769|WO2019011022A1|2017-07-14|2018-04-12|Application of cas protein, method for detecting target nucleic acid molecule and kit|

---