Use of protein cas, method for detection of nucleic acid molecule, and kit

Abstract

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 after the reaction is complete.

Description

1 / 60 USE OF CAS PROTEIN, METHOD FOR DETECTION OF NUCLEIC ACID MOLECULE, AND KIT TECHNICAL FIELD

[0001] The present invention belongs to the field of biotechnology and, in particular, to a method for detecting target nucleic acid molecules. FUNDAMENTALS OF THE INVENTION

[0002] A specific nucleic acid detection method has significant application value, such as pathogen detection, genetic disease detection, etc. In one aspect of pathogen detection, since each pathogenic microorganism has its own unique characteristic nucleic acid molecular sequence, it is possible to develop molecular nucleic acid detection for specific species, also known as nucleic acid diagnostics (NAD), which is of great importance in the areas of food safety, detection of environmental microorganism pollution, human pathogen infection, etc. Another aspect is the detection of single nucleotide polymorphisms (SNPs) 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.SNPs are closely related to biological functions, evolution, and disease; therefore, the development of SNP detection and analysis technologies is particularly important.

[0003] Currently, many NAD methods have been established, primarily for the detection of a specific DNA molecule, and there are also some methods for RNA molecules. Generally speaking, a DNA molecule is very stable; therefore, a test sample can come from a series of complex biological samples; RNA, however, degrades very easily and therefore needs to be handled with great care. In the 1970s, a detection method using restriction endonuclease digestion was established. Subsequently, methods such as Southern, Northern, and dot blot hybridization were developed for the specific detection of a nucleic acid molecule. In 1985, when PCR became a conventional experimental method, this led to an exponential improvement in molecular biology. The currently established specific nucleic acid molecule detection generally needs to be performed in two steps, the first step being the amplification of a target nucleic acid and the Petition 870200036677, dated 03 / 19 / 2020, p. 8 / 85 2 / 60 Second stage: detection of the target nucleic acid. PCR technology is an amplification method that was first established and is most commonly used today. Currently, based on the PCR method, a fluorescently labeled probe is introduced so that the amplification status 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), recombinant polymerase amplification (RPA), etc. The advantage of many of these alternative methods is isothermality.In other words, only one temperature is needed to complete the reaction, without the need for a thermal cycling instrument like the one used in PCR. Among nucleic acid detection methods, besides real-time PCR which can directly complete amplification and detection, FISH (fluorescence in situ hybridization) technology is the most commonly used detection method – a method in which a labeled molecular probe is hybridized in situ with a complementary target sequence. In addition, detection methods such as next-generation sequencing technologies and Oxford Nanopore sequencing technologies have also been developed, but these methods generally require expensive experimental equipment.

[0004] SNP detection also requires prior amplification by a method such as PCR and similar methods in order to obtain sufficient fragments of the SNP site-containing region for further detection. Commonly used methods include: primer extension, hybridization, ligation, and enzymatic cleavage. Once 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] Although many detection methods have been developed for the detection of nucleic acids, as described above, in certain cases, such as faster, simpler and more economical detection, it is still an important direction for development, such as the rapid detection of pathogenic bacteria in Petition 870200036677, dated 03 / 19 / 2020, p. 9 / 85 3 / 60 field, rapid detection of drug-sensitive SNPs, etc. In 2016, Collins et al. developed a rapid and inexpensive method for detecting the Zika virus based on the CRISPR-Cas9 characteristic of specifically recognizing and cleaving a target sequence. In 2017, Feng Zhang et al. established a rapid nucleic acid detection method 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 target RNA detection is achieved by combining it with an isothermal amplification technology by RPA, and Feng Zhang's team named this detection method SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking).The SHERLOCK method involves binding to an RNA template; therefore, if DNA detection is required, the DNA must first be transcribed into an RNA template for detection; and given the instability of RNA, this method will undoubtedly increase the degree of operational difficulty.

[0006] In 2015, Feng Zhang et al. discovered a novel CRISPR-related endoproteinase Cas12a (formerly known as Cpf1), which, like a commonly used Cas9 protein, is an RNA-guided DNA-specific 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] One objective of the present invention is to provide a method for detecting a nucleic acid target molecule.

[0008] Another objective of the present invention is to provide the use of a Cas protein in a method for detecting a nucleic acid target molecule.

[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] One method for detecting a target nucleic acid molecule includes adding a guide RNA, a Cas protein, a nucleic acid probe, and a Petition 870200036677, dated 03 / 19 / 2020, p. 10 / 85 4 / 60 buffer solution in a reaction system containing a target nucleic acid molecule to be detected, and then detect the target nucleic acid (especially by a fluorescence intensity detection method).

[0011] Preferably, the Cas protein is Cas12a or a Cas protein with single-stranded DNA cleavage collateral activity similar to Cas12a.

[0012] Preferably, the Cas protein is Cas12a.

[0013] Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.

[0014] Preferably, Cas12a is LbCas12a.

[0015] Preferably, guide RNA refers to an RNA that guides the Cas protein to bind specifically to a target DNA.

[0016] In another preferred embodiment, the nucleic acid probe is a single-stranded DNA; the single-stranded DNA is preferably a fluorescently labeled single-stranded DNA; the 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] 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] Preferably, the target nucleic acid molecule to be detected in the reaction system is obtained after amplification.

[0019] Preferably, the detection method of the present invention can be used to detect a specific pathogenic microorganism, genetic mutation, or target DNA.

[0020] In another preferred embodiment, the Cas protein includes Cas12b (C2c1).

[0021] In a second aspect of the present invention, the use of a Cas protein in a method for detecting a nucleic acid molecule is provided. Petition 870200036677, dated 03 / 19 / 2020, p. 11 / 85 5 / 60 target or in the preparation of a formulation to detect a target nucleic acid molecule.

[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] Preferably, guide RNA refers to an RNA that guides the Cas protein to bind specifically to a target DNA.

[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] In another preferred embodiment, the kit also includes a buffer solution.

[0026] In a fourth aspect of the present invention, a detection system is provided for detecting a target nucleic acid molecule, wherein the system includes: (a) a Cas protein, which is Cas12a or a Cas protein with collateral single-stranded DNA cleavage activity similar to Cas12a; (b) a guide RNA that guides the Cas protein to bind specifically to the target nucleic acid molecule; and (c) a nucleic acid probe that is single-stranded DNA; where the target nucleic acid molecule is a target DNA.

[0027] In another preferred embodiment, the detection system also includes (d) a buffer solution.

[0028] In another preferred embodiment, the detection system also includes a target nucleic acid molecule to be detected.

[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] In another preferred embodiment, in the detection system, the molar ratio of the nucleic acid probe to the target nucleic acid molecule is 103:1 to 1014:1 and, preferably, 104:1 to 107:1.

[0031] In another preferred embodiment, the detection site of the target nucleic acid molecule is located at positions 1-12 downstream of the sequence of Petition 870200036677, dated 03 / 19 / 2020, p. 12 / 85 6 / 60 PAM of the guide RNA.

[0032] In another preferred embodiment, the guide RNA length is 15-30 nt and preferably 15-18 nt.

[0033] In another preferred embodiment, the target DNA includes a cDNA.

[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] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a suppressor group.

[0036] In another preferred embodiment, the fluorescent group and the suppressor group are located independently at the 5' end, the 3' end, and the middle portion of the nucleic acid probe.

[0037] In another preferred embodiment, the nucleic acid probe length is 3-300 nt, preferably 5-100 nt, more preferably 6-50 nt, and even more preferably 8-20 nt.

[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] In another preferred embodiment, the target DNA is artificially synthesized or naturally occurring DNA.

[0040] In another preferred embodiment, the target DNA includes wild-type or mutant DNA.

[0041] In another preferred embodiment, the target DNA includes DNA obtained by reverse transcription of RNA or amplification, such as cDNA, etc.

[0042] In another preferred embodiment, Cas12a is selected from a group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; and more preferably, Cas12a is LbCas12a.

[0043] In another preferred embodiment, the Cas protein with a collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (i.e., C2c1). Petition 870200036677, dated 03 / 19 / 2020, p. 13 / 85 7 / 60

[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 (Alicyclobacillus macrosporangiidus), AhCas12b (Alicyclobacillus herbarius), and AcCas12b (Alicyclobacillus contaminans).

[0045] In another preferred embodiment, the nucleic acid probe comprises a single-stranded DNA carrying a detectable marker.

[0046] In another preferred embodiment, single-stranded DNA is single-stranded DNA labeled with fluorescence and labeled with biotin.

[0047] In another preferred embodiment, single-stranded DNA is single-stranded DNA labeled with fluorescence.

[0048] In another preferred embodiment, the single-stranded DNA is a fluorescent probe that is labeled with a fluorescent HEX group at a 5' terminus and is labeled with a BHQ1 suppressor group at a 3' terminus.

[0049] In a fifth aspect of the present invention, a kit is provided for detecting 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 a 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 bind specifically 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; where the target nucleic acid molecule is a target DNA.

[0050] In another preferred embodiment, any two, three or four (or all) of the first, second, third and fourth containers may be the same or different containers.

[0051] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a suppressor group.

[0052] In a sixth aspect of the present invention, a method is provided Petition 870200036677, dated 03 / 19 / 2020, p. 14 / 85 8 / 60 to detect whether a target nucleic acid molecule exists in a sample, including the following steps:

[0053] (a) provide the detection system for detecting a target nucleic acid molecule according to the fourth aspect of the present invention, wherein the detection system also has a sample to be detected; and

[0054] (b) detect whether the nucleic acid probe in the detection system is cleaved by a Cas protein, wherein the cleavage is a transcleavage of a collateral single-stranded DNA;

[0055] where 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] In another preferred embodiment, the sample to be detected includes an unamplified sample and an amplified (or nucleic acid-amplified) sample.

[0057] In another preferred embodiment, the sample to be detected is a sample obtained by amplification.

[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, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.

[0059] In another preferred embodiment, PCR includes high-temperature PCR, normal-temperature PCR, and low-temperature PCR.

[0060] In another preferred embodiment, the method is used to detect whether SNPs, point mutations, deletions and / or insertions exist in a nucleic acid at a target site.

[0061] In another preferred embodiment, 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] In another preferred embodiment, the PAM introducer primer has Petition 870200036677, dated 03 / 19 / 2020, p. 15 / 85 9 / 60 a formula I structure in 5'-3':

[0063] P1-P2-P3 (I)

[0064] in which,

[0065] P1 is a 5' segment sequence located at the 5' end and is complementary or non-complementary to the sequence of the target nucleic acid molecule;

[0066] P2 is a PAM sequence;

[0067] P3 is a 3' segment sequence located at the 3' end and is complementary to the sequence of the target nucleic acid molecule.

[0068] In another preferred embodiment, the PAM primer binds specifically upstream or downstream of the target nucleic acid molecule.

[0069] In another preferred embodiment, P1 has a length of 0-20 nt.

[0070] In another preferred embodiment, P3 has a length of 5-20 nt.

[0071] In another preferred embodiment, the PAM primer has a length of 18-50 nt, and preferably 20-35 nt.

[0072] In another preferred modality, the supplementation includes complete supplementation and partial supplementation.

[0073] In another preferred embodiment, at least one primer containing the PAM sequence is used in nucleic acid amplification.

[0074] In another preferred embodiment, 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 most preferably in the range of -10 nt to +10 nt) of the target site contains a PAM sequence, a primer containing or not containing the PAM sequence can be used and the amplified amplification product contains the PAM sequence.

[0075] In another preferred embodiment, detection in step (b) includes a fluorescence detection method.

[0076] In another preferred embodiment, the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer for detection.

[0077] In a seventh aspect of the present invention, use is provided of Petition 870200036677, dated 03 / 19 / 2020, p. 16 / 85 10 / 60 a Cas protein in the preparation of a reagent or detection kit to detect a target nucleic acid molecule based on single-stranded DNA cleavage, wherein the Cas protein is a Cas12a or Cas protein with a single-stranded DNA cleavage activity similar to that of Cas12a.

[0078] In another preferred embodiment, Cas12a is selected from a group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; and more preferably, Cas12a is LbCas12a.

[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] In another preferred embodiment, the Cas12b protein is selected from a group consisting of AacCas12b.

[0081] It should be understood that, within the scope of the present invention, the aforementioned technical features of the present invention and the technical features described in detail below (for example, in examples) can be combined to form a new or preferred technical solution. Due to length limitations, this will not be repeated here. BRIEF DESCRIPTION OF THE FIGURES

[0082] FIGURE 1 shows a c / s cleavage feature of Cas12a in the cleavage of a target single-stranded DNA.

[0083] FIGURE 2 shows that when cleaving a target single-stranded DNA, Cas12a does not depend on a PAM sequence required for the cleavage of double strands.

[0084] FIGURE 3 shows a Cas12a transcleavage feature in the cleavage of a target single-stranded DNA.

[0085] FIGURE 4 shows test Cas12as from 10 different sources, where all of these Cas12as have c / se cleavage activities and transcleavage in single-stranded DNA.

[0086] FIGURE 5 identifies sites possibly related to c / se transcleavage activities in single-stranded DNA in Cas12a through a single-site mutation experiment of Cas12a.

[0087] FIGURE 6 shows the structures of the Cas12a and monomers Petition 870200036677, dated 03 / 19 / 2020, page 17 / 85 11 / 60 Cas12b (i.e., C2c1) and its complexes with a guide RNA and a target DNA.

[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] FIGURE 8 shows a schematic flowchart of a HOLMES method for detecting a target DNA based on amplification of the target DNA and Cas12a transcleavage activity on a collateral single-stranded DNA.

[0090] FIGURE 9 shows a sensitivity test of a target DNA using FnCas12a or LbCas12a directly, or in combination with the HOLMES method.

[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 guide sequence lengths in combination with FnCas12a or LbCas12a.

[0092] FIGURE 11 tests whether a FAM-labeled single-stranded DNA probe is transcleaved after the addition of the target single-stranded DNA using a FAM-labeled fluorescent probe and 10 Cas12a proteins.

[0093] FIGURE 12 tests fluorescence values ​​after the addition of the target single-stranded DNA using HEX-N12-BHQ1 as a probe and 10 Cas12a proteins.

[0094] FIGURE 13 (A) shows the HOLMES detection values ​​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 single-stranded DNA fluorescent probe labeled with HEX and BHQ1 at both ends. It is shown that the fluorescence response value of Escherichia coli MG1655 decreases with decreasing concentration. (B) Detection values ​​of water samples in environments at different locations.

[0095] FIGURE 14 shows a schematic flowchart of a HOLMES method for detecting SNPs and fluorescence detection values ​​of 5 SNP sites.

[0096] FIGURE 15 shows the fluorescence detection values ​​of key sites in a TP53 gene (a cancer-related gene), as Petition 870200036677, dated 03 / 19 / 2020, p. 18 / 85 12 / 60 detected by the HOLMES method.

[0097] FIGURE 16 shows the detection values ​​of 5 SNP sites (related to the droplet), as detected by the HOLMES method.

[0098] FIGURE 17 shows the detection values ​​of an SNP (droplet-related) site, as detected by the HOLMES method, in which the samples are samples from 21 volunteers.

[0099] FIGURE 18 shows a primer design scheme of an example of the present invention, which can be used for SNP detection by HOLMES at any site.

[0100] FIGURE 19 uses a combination of LAMP and HOLMES to detect Escherichia coli in the system. (A) An electrophoresis map of an Escherichia coli gyrB gene amplified by LAMP. A total of two primer sets, 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 a LAMP amplification product. Negative control: the sample is sterile water and a gyrB-1 amplification primer is used to amplify or detect the gyrB gene output; gyrB-1: the sample is Escherichia coli to be detected and a first set of gyrB gene amplification primers is used to amplify or detect the gyrB gene output; In 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] FIGURE 20 detects the genotype of a human HEK293T cell using a combination of LAMP and HOLMES. (A) An electrophoresis map of a matching SNP detection model of the human HEK293T cell, as amplified by LAMP. Negative control: sample is sterile water and result is the result of amplification using an amplification primer rs5082; rs5082: sample is a total genome of the human HEK293T cell and result is the result of amplification using the amplification primer rs5082; and rs1467558: sample is the total genome of the human HEK293T cell and result is the result of amplification using an amplification primer rs1467558. (B) A HOLMES detection system is used to detect a LAMP amplification product. The rs5082 site was detected using two crRNAs, crRNA-G and crRNA-T, respectively (Sequence Listing 5); and the rs1467558 site was detected. Petition 870200036677, dated 03 / 19 / 2020, page 19 / 85 13 / 60 using two crRNAs of crRNA-C and crRNA-T, respectively, (Sequence Listing 5).

[0102] FIGURE 21 uses a combination of RPA and HOLMES to detect Escherichia coli in the system. (A) Amplification of the gyrB gene of Escherichia coli 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 amplification product of the RPA. Negative control: the sample is sterile water and a gyrB-1 amplification primer is used to amplify or detect the gyrB gene output; 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 gyrB gene output; In 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 gyrB gene result.

[0103] FIGURE 22 shows the detection of collateral single-stranded DNA cleavage activity by Cas12b when single-stranded DNA is used as the target DNA. After completion of the collateral cleavage reaction, the reagents are separated by electrophoresis on a 12% denatured urea 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 66 nt long single-stranded DNA at a dosage of 50 nM; and the single-stranded DNA probe is a single-stranded DNA carrying 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 the target DNA are contained, the FAM-labeled single-stranded DNA is cleaved into fragments, i.e., Cas12b has collateral single-stranded DNA cleavage activity.

[0104] FIGURE 23 shows the detection of collateral single-stranded DNA cleavage activity of Cas12b when a single-stranded DNA and a double-stranded DNA are used as target DNAs. After 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 66 nt long single-stranded DNA or double-stranded DNA at a dosage of 50 nM; and the single-stranded DNA probe is a single-stranded DNA probe (HEXN12-BHQ1) containing a fluorescence reporter group and a suppressor group in Petition 870200036677, dated 03 / 19 / 2020, page 20 / 85 14 / 60 dosage of 500 nM. As can be seen in the figure, regardless of whether a single-stranded DNA template or a double-stranded DNA template is used, the cleavage activity of collateral single-stranded DNA can be detected after the addition of Cas12b and guide RNA.

[0105] FIGURE 24 shows the transcleavage activity of Cas12b on collateral single-stranded DNA in a low-concentration target DNA after combination with LAMP amplification. DETAILED DESCRIPTION

[0106] In order to make clearer the objectives, technical solutions and advantages of the embodiments of the present invention, the following clearly and completely describes the technical solutions in the embodiments of the present invention with reference to the drawings accompanying the embodiments of the present invention. The embodiments described are a part and not all embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative effort fall within the scope of protection of the present invention.

[0107] The inventor has developed a technical solution for the detection of target nucleic acid through extensive and in-depth research and investigation into the cleavage characteristics of a Cas enzyme (such as Cas12a and Cas12b enzymes). Experimental results show that a nucleic acid is successfully and rapidly detected by employing the aforementioned technical solution, for example, to identify whether a certain concentration of microorganisms such as Escherichia coli is present in water and to rapidly identify an SNP genotype. The present invention is concluded on this basis. Terms

[0108] The term guide RNA refers to an RNA that guides a Cas protein to bind specifically to a target DNA sequence.

[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] The term CRISPR refers to a clustered regularly interspaced short palindromic repeat, which is the immune system of many prokaryotes.

[0111] The term Cas protein refers to a protein associated with Petition 870200036677, dated 03 / 19 / 2020, p. 21 / 85 15 / 60 CRISPR, which is a related protein in a CRISPR system.

[0112] The term Cas12a (formerly known as Cpf1) refers to a crRNA-dependent endonuclease, which is a VA-type enzyme in the CRISPR system classification.

[0113] The terms Cas12b and C2c1 are used interchangeably and refer to a crRNA-dependent endonuclease, which is a VB-type enzyme in the CRISPR system classification.

[0114] The term LAMP is a loop-mediated isothermal amplification technology, which is an isothermal nucleic acid amplification technology suitable for gene diagnostics.

[0115] The term PAM refers to a motif adjacent to the protospacer, necessary for Cas12a splitting. FnCas12a PAM is a TTN sequence, LbCas12a PAM is a TTTN sequence, and AacCas12b PAM is TTN.

[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 to a reaction system containing a target nucleic acid molecule to be detected and then performing fluorescence detection of the target nucleic acid molecule.

[0117] The Cas protein is Cas12a or Cas12b.

[0118] Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; and Cas12a is preferably LbCas12a.

[0119] Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b or AcCas12b.

[0120] Guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.

[0121] The target nucleic acid molecule to be detected in the reaction system is obtained by amplification.

[0122] The detection method can detect a pathogenic microorganism, genetic mutation, or a specific target DNA.

[0123] Use of a Cas protein in a method to detect a Petition 870200036677, dated 03 / 19 / 2020, p. 22 / 85 16 / 60 target nucleic acid molecule.

[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] Guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.

[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 may also include a buffer solution.

[0127] The present invention provides a detection method for rapidly detecting a target nucleic acid molecule with high specificity. When the 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 design, the crRNA targets the target DNA (a segment of the DNA sequence to be detected); crRNA and Cas12a protein are added to the detection system; when the target DNA is present, Cas12a forms a ternary complex with the crRNA and the target DNA, and while this is happening, the complex exerts its collateral cleavage activity and cleaves a single-stranded DNA labeled with a fluorescent signal (two ends of the single-stranded DNA 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 is possible to determine 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 rapidly 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] 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] Cas12a selection: according to the research, Cas12a has a Petition 870200036677, dated 03 / 19 / 2020, p. 23 / 85 17 / 60 transcleavage activity, that is, once the target DNA, crRNA, and Cas12a protein form a ternary complex, other single-stranded DNAs (collateral single-stranded DNAs) 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 random 12 nt sequence, and is labeled with a fluorescent HEX group at the 5' end and a BHQ1 suppressor group (HEX-N12-BHQ1) at the 3' end. When the target DNA fragment is contained in the system, a ternary complex of the target DNA, crRNA, and Cas12a protein will be formed. At this point, the probe will be cleaved, and meanwhile, the fluorescent HEX 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 a double-stranded DNA, as shown in FIGURE 7.It can be observed that the complex composed of the target double-stranded DNA and each Cas12a protein can perform transcleavage activity.

[0130] HOLMES response sensitivity: Next, the response sensitivities of FnCas12a and LbCas12a to the target DNA are tested, i.e., the lowest concentration of the target DNA at 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, i.e., amplification of the fragment of interest via PCR followed by a Cas12a cleavage reaction, the response sensitivity can be as low as 10 aM, as shown in FIGURE 9.

[0131] SNP test: Next, it is tested whether the HOLMES method can detect an SNP genotype. T1 is used as a target sequence, PAM at this site is mutated or positions 1-18 of the target sequence are respectively subjected to single point mutation, and the detection differences between an unmutated sequence and a mutated sequence by crRNAs of different lengths are compared.

[0132] As shown in FIGURE 10, when the complementary target sequence is a 24 nt crRNA (crRNA-24nt), the single point mutation at the positions Petition 870200036677, dated 03 / 19 / 2020, p. 24 / 85 18 / 60 8-18 is not very different from the wild type, while the fluorescence value obviously decreases after PAM mutation and mutation of positions 1-7. When crRNA is truncated and the length of a paired target sequence is 18 nt, the fluorescence value of the mutation positions from 8-16 nt is obviously decreased compared to that of a target sequence with a length of 24 nt; when the crRNA length continues to be reduced to 16 nt or 17 nt, the fluorescence value of the mutated target sequence decreases more significantly; and when it is further reduced to 15 nt, the fluorescence value of the mutated target sequence is weaker compared to that of this target sequence, but the fluorescence intensity of the mutated target sequence may still be higher compared to those of other target sequences and therefore can be used for detection. Taken together, the 15 nt, 16 nt, and 17 nt crRNAs are the most suitable for SNP detection.

[0133] In the present invention, Cas12a cleaves single-stranded DNA through a form of sequence-independent programmed cleavage PAM, which is called cis cleavage; but once the ternary Cas12a / crRNA / target DNA complex is formed, it will exhibit transcleavage activity, i.e., it will exhibit cleavage activity of any non-target single-stranded DNA in the system.

[0134] Based on the characteristics of Cas12a, a method for specific detection of nucleic acid molecules is developed, called HOLMES (one Hour Lowcost Multipurpose Efficient Simple assay). As the name suggests, the technology is characterized by a fast (1 hour), low-cost, multi-channel, highly efficient, and simple testing method. The method can be used in the fields of rapid pathogen detection, SNP detection, and similar applications. Nucleic acid detection based on collateral cleavage activity

[0135] The present invention also provides a nucleic acid detection method based on the collateral cleavage activity of the Cas12 enzyme (including Cas12a or Cas12b).

[0136] Preferably, detection of the present invention can be performed for SNP and, in particular, PCR amplification is performed first, Petition 870200036677, dated 03 / 19 / 2020, p. 25 / 85 19 / 60 followed by detection.

[0137] With reference to FIGURE 18, a primer design scheme is provided.

[0138] Case 1. When a PAM site exists near 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] 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] A typical step involves designing a primer near the SNP site and loading a PAM site onto the primer, where the sequence located at the 3' end of the PAM site must be paired with the template DNA. There is no special requirement for the primer at the other end, as long as the primer can be paired with the template DNA and can be used for PCR amplification. As shown in FIGURE 18, PAM sites can be successfully introduced after PCR amplification.

[0141] With reference to FIGURE 10, in the present invention, when designing the introduction of the PAM site, the SNP site is generally located in the first 16 bases of the 5' terminal of the crRNA guide sequence, preferably at positions 1-14, more preferably at positions 1-12, even more preferably at positions 1-11 or 1-10, and much more preferably at positions 1-8 or 1-7.

[0142] The present invention has the following main advantages:

[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] (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] (3) High efficiency: the present invention has sensitivity Petition 870200036677, dated 03 / 19 / 2020, p. 26 / 85 20 / 60 extremely high and can detect DNA at a concentration of 10 aM.

[0146] (4) Multiple uses: can detect different nucleic acid samples, including DNA and RNA samples.

[0147] (5) Simplicity: there are no special or complicated steps and, if a kit is prepared and a program is set, only simple operations are required, such as adding a sample.

[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 limiting the scope of the present invention. The experimental methods in the following examples, which are not specified with specific conditions, are generally carried out 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 indicated, percentages and parts are weight percentages and parts by weight.

[0149] Unless specifically indicated otherwise, the experimental materials involved in the present invention can be obtained from commercial channels. Materials

[0150] 1. The RNase inhibitor is purchased from TaKaRa and the high-fidelity DNA polymerase KOD FX is purchased from ToYoBo; the primers (oligonucleotides) are synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the 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 cleanup and PCR system is purchased from Promega; and all media (e.g., tryptone, yeast extract, etc.) are purchased from OXOID.

[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 a target single-stranded DNA (the probe being labeled with FAM).

[0152] A single-stranded DNA (T1-R target) was selected as the target sequence to test the response values ​​of its detection by different Cas12a proteins. Petition 870200036677, dated 03 / 19 / 2020, p. 27 / 85 21 / 60

[0153] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7T1-24-R, as shown in Table 5. Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0154] 2. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, a Cas12a (0.25 pM), a target single-stranded DNA (T1-R target) (0.01 pM), a nucleic acid probe (N25-5 ΈAM) (0.01 pM), a NEB 3.1 buffer solution, and 0.5 pL 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 terminated at 98 °C for 2 min.

[0155] 3. Fluorescence detection: the reaction was subjected to urea-acrylamide gel electrophoresis (Urea-PAGE) and then detected with a fluorescence luminescence imager. As shown in FIGURE 11, different Cas12as have different detection effects on the target. For example, for HkCas12a etc., probe cleavage was caused even when no target single-stranded DNA was added. LbCas12a and similar are better candidates for Cas12a proteins because probe cleavage occurred only when target single-stranded DNA was added. Example 2: Detection of Cas12a protein capable of detecting a target single-stranded DNA (the probe being labeled with two markers, HEX and BHQ1).

[0156] A single-stranded DNA (T1-R target) was selected as the target sequence to test the response values ​​of its detection by different Cas12a proteins. Petition 870200036677, dated 03 / 19 / 2020, p. 28 / 85 22 / 60

[0157] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0158] 2. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, a Cas12a (0.25 pM), a target single-stranded DNA (T1-R target) (0.01 pM), a fluorescence probe (HEX-N12BHQ1, i.e., a 12 nt single-stranded DNA labeled with HEX at the 5' end and BHQ1 at the 3' end) (0.5 pM), a NEB 3.1 buffer solution, and 0.5 pL 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 terminated at 98 °C for 2 min.

[0159] 3. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 Cas12as have different detection effects on the target. For example, for HkCas12a etc., probe cleavage was caused even when no target single-stranded DNA was added. FnCas12a and similar are better candidates for Cas12a proteins because probe cleavage occurred only when target single-stranded DNA was added. Example 3: Detection of Cas12a protein capable of detecting a target double-stranded DNA.

[0160] A double-stranded DNA (T1 target) was selected as the target sequence to test the response values ​​of its detection by different proteins. Petition 870200036677, dated 03 / 19 / 2020, p. 29 / 85 23 / 60 Cas12a.

[0161] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0162] 2. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, a Cas12a (0.25 pM), a target double-stranded DNA (T1 target, obtained by annealing T1-F target primers to T1-R target) (0.01 pM), a fluorescence probe (HEX-N12-BHQ1) (0.5 pM), a NEB 3.1 buffer solution and 0.5 pL of an RNase inhibitor. The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0163] 3. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 similar are better candidates for Cas12a proteins because probe cleavage occurred only when the target double-stranded DNA was added. Example 4: Testing different target concentrations with FnCas12a and LbCas12a.

[0164] T1 target was selected as the target DNA and then diluted at different concentrations in a gradient to test the sensitivity of the FnCas12a and LbCas12a response to it. To increase sensitivity, a PCR amplification step was added.

[0165] 1. crRNA preparation: first, a transcript template was prepared by annealing T7-crRNA-F to a synthesized T7 oligonucleotide. Petition 870200036677, dated 03 / 19 / 2020, p. 30 / 85 24 / 60 T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by cooling from 95 °C to 20 °C at a rate of 1 °C per minute using a thermocycler. The crRNA was synthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was then purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0166] 2. PCR Amplification (Optional): A plasmid containing the T1 target (pUC18-T1) was used as a template, diluted in a gradient, and then used for the PCR reaction. The total volume of each reaction system was 20 pL, 0.25 pM of M13F-47 and M13R-48 were used as primers (Table 4), and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was at 95 °C for 2 min, followed by 35 cycles at 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 10 s. After completion of the PCR, the PCR amplification product was used directly for a Cas12a reaction.

[0167] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, FnCas12a or LbCas12a (0.25 pM), 1 pL of a PCR product (or target DNAs that are directly diluted to different concentrations), a fluorescent probe (HEX-N12-BHQ1) (0.5 pM), a NEB 3.1 buffer solution, and 0.5 pL of an RNase inhibitor. The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0168] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 concentrations above 0.1 nM could respond, and the response was notable when the concentration was above 1 nM. If a PCR technology was combined, i.e., 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 aM. Petition 870200036677, dated 03 / 19 / 2020, p. 31 / 85 25 / 60 Example 5 Testing the single-point mutant target with FnCas12a and LbCas12a

[0169] 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 several crRNAs of different lengths for the wild type and the same after single point mutation.

[0170] 1. crRNA preparation: first, a transcript template 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 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL 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 T7 high-throughput transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was then purified using an RNA purification and concentration kit, quantified with a NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0171] 2. PCR Amplification: A plasmid containing the T1 target (pUC18-T1) was used as a template. The total volume of each reaction system was 20 pL, 0.25 pM of the M13R-48 primer and each respective mutation primer for the T1F target was used (Table 4), and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was carried out at 95 °C for 2 minutes and then initiated 35 cycles of 98 °C for 10 s, 60 °C for 15 s and 68 °C for 10 s. After completion of PCR, the product was used directly for a Cas12a reaction.

[0172] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, FnCas12a or LbCas12a (0.25 pM), 1 pL of a PCR product, a fluorescent probe (HEX-N12-BHQ1) (0.5 pM), a NEB 3.1 buffer solution, and 0.5 pL of an RNase inhibitor. The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0173] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were added to a 96-well plate and then detected by a reader. Petition 870200036677, dated 03 / 19 / 2020, page 32 / 85 26 / 60 microplates (with an excitation light at 535 nm and an emission light at 556 nm). As shown in FIGURE 10, when the complementary target sequence was a 24 nt crRNA (crRNA-24nt), single point mutations at positions 818 were not very different from the wild type, while the fluorescence value decreased obviously after PAM mutation and point mutation at positions 17.When the crRNA was truncated and the length of a paired target sequence was 18 nt, the fluorescence value of the 816 nt mutation positions decreased noticeably compared to that of 24 nt; when the length was 16 nt or 17 nt, the decrease in the fluorescence value of the mutated target sequence was more obvious; and when the length was 15 nt, the fluorescence values ​​of both the target sequence and the mutated target sequence were very weak, but the fluorescence intensity of the mutated target sequence could still be higher compared to those of other target sequences and therefore could be used for detection. Taken together, the 15 nt, 16 nt, and 17 nt crRNAs are the most suitable for SNP detection. Example 6: Testing for Escherichia coli and similar microorganisms in ambient water.

[0174] The gyrB gene of Escherichia coli 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 water (such as sewage and tap water) in the environment was determined.

[0175] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C. Petition 870200036677, dated 03 / 19 / 2020, page 33 / 85 27 / 60

[0176] 2. PCR Amplification: When the positive control sample Escherichia coli MG1655 was cultured until ODeoo reached approximately 0.5, it was diluted in a 10-fold gradient, respectively, and then used as a template, and the sample was ambient water (including tap water and muddy ambient water). The total volume of each reaction system was 20 pL, 0.25 pM of each of the primers gyrB-F and gyrB-R that were used (Table 4), and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was carried out at 95 °C for 2 minutes and then initiated 35 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 10 s. After the PCR was complete, the PCR product was used directly for a Cas12a reaction.

[0177] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, LbCas12a (0.25 pM), 1 pL of a PCR product, a fluorescence probe (HEX-N12-BHQ1) (0.5 pM), a NEB 3.1 buffer solution, and 0.5 pL of an RNase inhibitor. The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0178] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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, the microorganisms were detected most obviously in samples 2, 4, 5, and 6. Example 7 Human SNP Test

[0179] The SNP test selected 5 human SNP sites, namely rs5082, rs1467558, rs2952768, rs4363657 and rs601338, to test the feasibility of the HOLMES method.

[0180] 1. crRNA Preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 pM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by 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-throughput T7 transcript kit, and the reaction was carried out overnight at 37 °C. Petition 870200036677, dated 03 / 19 / 2020, p. 34 / 85 28 / 60 (for approximately 16 h). The RNA was purified using the RNA Clean & Concentrator™-5 (Zymo Research), quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0181] 2. PCR Amplification: The total volume of the reaction system was 20 pL. 0.25 pM of each primer was used (Table 4). 1 ng of a human genome (HEK293T) or directly scraped oral epithelial mucosa was used as a template, and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was performed at 95 °C for 2 minutes, followed by 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-FT, 2-rs1467558-FT, and 3-rs2952768-RC were introduced directly into the corresponding SNP mutation products.)

[0182] 3. Cas12a reaction: a 20 pL reaction system was added with crRNA (1 pM), corresponding LbCas12a (0.5 pM), 1 pL of a PCR product and a fluorescent probe (HEX-N12-BHQ1) (0.5 pM). The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0183] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 matched the corresponding target sequence was there a higher fluorescence response value, and if there was a single point mutation, its response value would be significantly reduced. The corresponding SNP genotype can be determined by the fluorescence value, and these results were confirmed by sequencing results. Example 8: Testing a gene related to cancer.

[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 gene inactivation. A cell with a normal gene at this site (HEK293T), an individual gene, and a mutant T24 cell were tested, respectively.

[0185] 1. crRNA preparation: first, a transcript template was prepared by annealing T7-crRNA-F to the synthesized oligonucleotides T7crRNA-34-TP53-T24-C-16nt and T7-crRNA-34-TP53-T24-G-16nt (Table 5). Petition 870200036677, dated 03 / 19 / 2020, page 35 / 85 29 / 60 Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by cooling from 95 °C to 20 °C at a rate of 1 °C per minute using a thermocycler. The crRNA was synthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was purified using the RNA Clean & Concentrator™-5 (Zymo Research), quantified with NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80 °C.

[0186] 2. PCR Amplification: The total volume of the reaction system was 20 pL. 0.25 pM of each primer 34-TP53-T24-F and 34-TP53-T24-R (Table 4) were used, 1 ng of a human genome (HEK293T, T24) or directly scraped oral epithelial mucosa as a template, and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was carried out at 95 °C for 2 minutes and then initiated 35 cycles of 98 °C for 10 s, 60 °C for 15 s and 68 °C for 10 s. After completion of PCR, the product was used directly for a Cas12a reaction.

[0187] 3. Cas12a reaction: a 20 pL reaction system was added with crRNA (1 pM), corresponding LbCas12a (0.5 pM), 1 pL of a PCR product and a fluorescent probe (HEX-N12-BHQ1) (0.5 pM). The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0188] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 template, the detected value of crRNA-C was significantly higher than that of crRNA-G, while the crRNA-G of the T24 mutant cell was significantly increased. Example 9 Human SNP test (genes related to gout)

[0189] The SNP test selected 5 human SNP sites that were associated with gout risk, such as rs1014290, rs6449213, rs737267, rs1260326 and rs642803, to test the HOLMES method.

[0190] 1. crRNA preparation: first, a transcription template was Petition 870200036677, dated 03 / 19 / 2020, p. 36 / 85 30 / 60 prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was purified using the RNA Clean & Concentrator™5 (Zymo Research), quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C.

[0191] 2. PCR Amplification: The total volume of the reaction system was 20 pL, 0.25 pM of each primer was used (Table 4), 1 ng of a human genome (HEK293T) or directly scraped oral epithelial mucosa was used as a template, and a high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was carried out at 95 °C for 2 minutes and then initiated 35 cycles of 98 °C for 10 s, 60 °C for 15 s and 68 °C for 10 s. After completion of PCR, the product was used directly for a Cas12a reaction. (Primers 1-rs5082-FT, 2-rs1467558-FT and 3-rs2952768-RC were introduced directly into the corresponding SNP mutation products)

[0192] 3. Cas12a reaction: a 20 pL reaction system was added with crRNA (1 pM), corresponding LbCas12a (0.5 pM), 1 pL of a PCR product and a fluorescence probe (HEX-N12-BHQ1) (0.5 pM). The reaction was carried out at 37 °C for 15 min and then stopped at 98 °C for 2 minutes.

[0193] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 matched the corresponding target sequence was there a higher fluorescence response value, and if there was a single point mutation, its response value would be significantly reduced. The corresponding SNP genotype could be determined by the fluorescence value, and these results were confirmed by sequencing results. Example 10 SNP test of clinical samples from volunteers (one gene) Petition 870200036677, dated 03 / 19 / 2020, p. 37 / 85 31 / 60 related to gout) through a kit

[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 associated with gout risk.

[0195] 1. Kit Preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL and then subjected to an annealing procedure: initial denaturation at 95 °C for 5 minutes, followed by 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-throughput T7 transcript kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was purified using the RNA Clean & Concentrator™5 (Zymo Research), quantified with NanoDrop 2000C, and diluted to a concentration of 10 pM.

[0196] 2. Premixing in a 96-well plate for PCR: a 19 pL system was added with the reagents required for a PCR reaction, namely primers 41-rs1014290-F and 41-rs1014290-R.

[0197] 3. Premixing in a 96-well plate for fluorescence detection: a crRNA (1 pM), LbCas12a (0.5 pM) and a fluorescence probe (HEX-N12-BHQ1) (0.5 pM) were added to the 19 pL system and it was added to the 96-well plate.

[0198] 4. PCR amplification: the genomic DNAs of the volunteers were added to the premixed 96-well PCR plate and then subjected to the PCR reaction. The PCR reaction procedure was carried out at 95 °C for 2 minutes and then initiated 35 cycles at 98 °C for 10 s, 60 °C for 15 s and 68 °C for 10 s.

[0199] 5. Cas12a reaction: 1 pL of a PCR reaction solution was taken and added to the premixed 96-well plate for fluorescence detection, reacted at 37 °C for 15 minutes, and then the reaction was stopped at 98 °C for 2 minutes.

[0200] 6. Fluorescence detection: was detected by a reader of Petition 870200036677, dated 03 / 19 / 2020, p. 38 / 85 32 / 60 microplates (with an excitation light of 535 nm and an emission light of 556 nm). As shown in FIGURE 17, since the population with the A:A genotype had a higher risk of having gout, other people besides volunteers no. 5, 7 and 9 are of the A:G or G:G genotype; therefore, more attention should be paid to the risk of gout. Example 11 Detection of Escherichia coii and similar microorganisms in ambient water by LAMP combined with a Cas protein

[0201] The gyrB gene of Escherichia coii was selected as a detection target to indirectly test whether concentrations of Escherichia coii and similar microorganisms existed in the water.

[0202] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X Taq DNA polymerase reaction buffer (Transgen Biotech) to a total volume of 50 pL 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, finally diluted to a concentration of 10 pM and stored in a refrigerator at -80 °C for later use.

[0203] 2. LAMP Amplification: Sterile water and a contaminated liquid containing Escherichia coii were used as a negative control and as the sample to be detected, respectively. The total volume of each reaction system was 25 pL, using primers of 1.6 pM LAMP-FIP and LAMP-BIP each, 0.2 pM LAMP-F3 and LAMP-B3 each, 0.4 pM 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 performed at 65 °C for 30 minutes. After completion of LAMP, annealing was performed at 80 °C for 10 minutes, and then the product was used directly for a Cas12a reaction.

[0204] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, Cas12a (0.25 pM), 1 pL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1) (0.5 pM), a solution Petition 870200036677, dated 03 / 19 / 2020, page 39 / 85 33 / 60 buffer NEB 3.1 and 0.5 µL of an RNase inhibitor. The reaction was conducted at 37 °C for 15 minutes.

[0205] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 SNPs using LAMP amplification combined with a Cas protein

[0206] 1. crRNA preparation: first, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7crRNA-rs5082-T / T7-crRNA-rs5082-G / T7-crRNA-rs1467558-T / T7-crRNA-rs1467558-C (Table 5). Specifically, the paired oligonucleotide (4 pM) was annealed in a 1X Taq DNA polymerase reaction buffer (Transgen Biotech) to a total volume of 50 pL 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 T7 high-throughput transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). The RNA was then purified using an RNA purification and concentration kit, quantified with a NanoDrop 2000C, finally diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C for later use.

[0207] 2. LAMP Amplification: A human HEK293T genome was used as a sample. The total volume of each reaction system was 25 pL. Primers of 1.6 pM LAMP-FIP and LAMP-BIP each, 0.2 pM LAMP-F3 and LAMP-B3 each, 0.4 pM LAMP-LoopF ​​and LAMP-LoopB each were used, and the WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. The LAMP reaction procedure was performed at 65 °C for 30 minutes. After LAMP completion, annealing was performed at 80 °C for 10 minutes, and then the product was used directly for a Cas12a reaction.

[0208] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, Cas12a (0.25 pM), 1 pL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1) (0.5 pM), a NEB 3.1 buffer solution, and 0.5 pL of an RNase inhibitor. The reaction was Petition 870200036677, dated 03 / 19 / 2020, page 40 / 85 34 / 60 cooked at 37°C for 15 minutes.

[0209] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 20. Example 13 Detection of Escherichia coii and similar microorganisms in ambient water by RPA amplification combined with a Cas protein

[0210] The gyrB gene of Escherichia coii was selected as a detection target to indirectly test whether concentrations of Escherichia coii and similar microorganisms existed in the water.

[0211] 1. crRNA preparation: First, a transcript template was prepared by annealing T7-crRNA-F to a synthesized oligonucleotide T7crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 50 pL 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-throughput T7 transcription kit, and the reaction was carried out overnight at 37 °C (for approximately 16 h). Next, the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop2000C, finally diluted to a concentration of 10 pM and stored in a refrigerator at -80 °C for later use.

[0212] 2. RPA Amplification: Sterile water and a contaminated liquid containing Escherichia coii were used as a negative control and as a sample to be detected, respectively. The total volume of each reaction system was 25 pL, 0.5 pM of each primer RPA-gyrB-F (or RPA-gyrB-F2) and RPA-gyrB-R2 was used, and a TwistAmp® Basic (TwistDX) kit was used for the RPA reaction. The RPA reaction procedure was carried out at 37 °C for 30 minutes. After RPA completion, annealing was performed at 80 °C for 10 minutes, and then the product was used directly for a Cas12a reaction.

[0213] 3. Cas12a reaction: a 20 pL reaction system was added with the purified crRNA (0.5 pM) from step 1, Cas12a (0.25 pM), and 1 pL of a product. Petition 870200036677, dated 03 / 19 / 2020, page 41 / 85 35 / 60 of RPA, a fluorescence probe (HEX-N12-BHQ1) (0.5 μM), a NEB buffer solution 3.1 and 0.5 μL of an RNase inhibitor. The reaction was conducted at 37 °C for 15 minutes.

[0214] 4. Fluorescence detection: 20 pL of the inactivated reaction solution were 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 21. Example 14: Cas12b having a collateral dipping activity

[0215] 1. Preparation of a guide RNA (sgRNA)

[0216] First, a pUC18 guide RNA-T1 plasmid was constructed using pUC18 as the plasmid backbone. In the plasmid, a T7 promoter and a template DNA sequence for guide RNA transcription were inserted into pUC18 (Note: the guide RNA transcribed from this template was directed to a sequence called T1 in this study). The method first performed a round of PCR using the pUC18 plasmid as a template and PUC18-1-F and pUC18-1R as primers; ligating PCR products with a T4 DNA ligase, transforming the product into DH10b, and sequencing to obtain the correct clone, which was called the pUC18 guide RNA-T1-pre. Next, a second round of PCR was performed using pUC18 guide RNA-T1-pre as a template and pUC18-2-F and pUC18-2R as primers, ligating and transforming the PCR products in the same manner, to finally obtain the pUC18 guide RNA-T1 plasmid that was correct as sequenced.

[0217] Next, using the pUC18 guide RNA-T1 plasmid as a template, a guide RNA was synthesized using a high-throughput T7 transcription kit (Thermo), and the reaction was carried out overnight at 37 °C (for 12-16 hours).

[0218] Finally, DNase I (2 pL of DNase I were added per 50 pL of the transcription system) was added to the transcription system, the system was placed in a water bath at 37 °C for 30 minutes to eliminate plasmid DNA, and the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, diluted to a concentration of 10 pM, and stored in a refrigerator at -80 °C for later use.

[0219] 2. Preparation of a target DNA

[0220] (1) If the target DNA is single-stranded, a 66 bp oligonucleotide Petition 870200036677, dated 03 / 19 / 2020, page 42 / 85 36 / 60 of length is directly synthesized as the target DNA (T1-R target), which contained the 20 bp target sequence (T1) recognized by the guide RNA.

[0221] (2) If the target DNA is double-stranded, two complementary 66 bp long oligonucleotides (T1-F target; -T1-R target) are directly synthesized, containing the 20 bp target sequence (T1) identified by the guide RNA. The two oligonucleotides were annealed to obtain a short target DNA. Specifically, the paired oligonucleotide (1 μM) was annealed in 1X PCR buffer (Transgen Biotech) to a total volume of 20 pL 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] 3. Cas12b reaction

[0223] (1) Guide RNA annealing: the guide RNA was diluted to an appropriate concentration (10 pM) and annealed in 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] (2) Incubation of guide RNA with C2c1: the annealed guide RNA was mixed with C2c1 at an equal molar concentration and left to stand at 30 °C for 20-30 minutes.

[0225] (3) Cas12b reaction: a 20 pL reaction system was added with a mixture of guide RNA and C2c1 incubated in step (2) (both final concentrations were 250 pM or 500 pM), a target DNA (with a final concentration of 50 nM), a FAM-labeled oligonucleotide (DNMT1-3R-FAM-5' target) or a fluorescence quenching probe (HEX-N12-BHQ1 with a final concentration of 500 nM), 2 pL of 10 X NEB 3,1 buffer and 0.5 pL of an RNase inhibitor (40 U / pL). After mixing homogeneously, the reaction was conducted at 48 °C for 30 minutes. After that, it was inactivated by heating at 98 °C for 5 minutes in a PCR instrument.

[0226] 4. Detection of Cas12b transcleavage activity by denatured urea gel electrophoresis: 20 pL of the inactivated reaction solution were separated by a denatured urea gel electrophoresis method and then imaged using an ImageQuant LAS 4000 mini fluorescence imaging system (GE Healthcare). The results were as follows: Petition 870200036677, dated 03 / 19 / 2020, page 43 / 85 37 / 60 shown in FIGURE 22.

[0227] 5. Detection of Cas12b transcleavage activity by a fluorescence microplate reader method: 20 pL of the inactivated reaction solution were 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). The results were as shown in FIGURE 23. Example 15: Sensitivity test of a Cas12b reaction (transcleavage)

[0228] By detecting the excited fluorescence intensity of the fluorescence probe (HEX-N12-BHQ1), the target DNA concentration required for Cas12b to exert transcleavage activity, i.e., the sensitivity of a Cas12b transcleavage reaction, was determined.

[0229] 1. Preparation of a guide RNA

[0230] First, using the pUC18 guide RNA-T1 as a template and the DNMT1-3-F guide RNA and the DNMT1-3-R guide RNA as primers, 20 bases of the guide RNA targeting the T1 target DNA were replaced by a DNMT1-3-directed guide RNA by PCR, in order to obtain another pUC18 guide RNA-DNMT1-3 plasmid.

[0231] Next, using the pUC18 guide RNA-DNMT1-3 plasmid as a template, a guide RNA was synthesized using a high-throughput T7 transcription kit (Thermo), and the reaction was carried out overnight at 37 °C (for 12-16 hours).

[0232] Finally, DNase I (2 pL of DNase I were added per 50 pL of the transcription system) was added to the transcription system, the system was placed in a water bath at 37 °C for 30 minutes to eliminate plasmid DNA, and the RNA was purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C, and stored in a refrigerator at -80 °C for later use.

[0233] 2. Preparation of a target DNA

[0234] For the target DNA, the target DNA was first added directly to the Cas12b reaction system without amplification. The method was as follows:

[0235] (1) If the target DNA is single-stranded, a 50 bp long oligonucleotide is directly synthesized as the target DNA (DNMT 1 -3 (TTC PAM) Petition 870200036677, dated 03 / 19 / 2020, page 44 / 85 38 / 60 R), which contained the 20 bp target sequence (DNMT1-3) recognized by the guide RNA.

[0236] (2) If the target DNA is double-stranded, two complementary 50 bp long oligonucleotides (DNMT1-3 (TTC PAM)-F; DNMT1-3 (TTC PAM)-R) are directly synthesized, containing the 20 bp target sequence (DNMT 1-3) 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 1X PCR buffer (Transgen Biotech) to a total volume of 20 pL 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 thermocycler.

[0237] (3) The single- or double-stranded target DNA was gradually diluted to 2 pM, 0.2 pM, 0.02 pM, 0.002 pM and 0.0002 pM for further use.

[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] (1) The fragment containing the target sequence (DNMT1-3) was inserted into a pEasy-Blunt zero-cloning vector using the Transgen pEasyBlunt zero-cloning kit, in order to obtain the correct clone after sequencing verification.

[0240] (2) A LAMP expansion reaction

[0241] Using the above plasmid as a template, the LAMP amplification reaction was performed. Templates were added at 0 nM, 1 nM, and 0.1 nM respectively and diluted with a 10-fold gradient to 10'11nM. The total volume of each reaction system was 25 pL, using primers of 1.6 pM LAMP-DNM-FIP and LAMP-DNM-BIP each, 0.2 pM LAMP-DNM-F3 and LAMP-DNMB3 each, 0.4 pM 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 performed at 65 °C for 30 minutes. After the LAMP process was completed, inactivation was performed at 80 °C for 10 minutes, and then the product was used directly for a Cas12b reaction.

[0242] 3. Cas12b reaction

[0243] (1) Guide RNA annealing: the guide RNA was diluted to an appropriate concentration (5 pM) and annealed in the PCR instrument. The annealing procedure: denaturation at 75 °C for 5 minutes and then Petition 870200036677, dated 03 / 19 / 2020, p. 45 / 85 39 / 60 cooling from 75 °C to 20 °C at a decreasing rate of 1 °C per minute.

[0244] (2) guide RNA incubation with Cas12b: the annealed guide RNA was mixed with Cas12b at an equal molar concentration and left to stand at 30 °C for 20-30 minutes.

[0245] (3) Cas12b reaction: a 20 pL reaction system was added with the guide RNA and Cas12b mixture incubated in step (2) (both final concentrations of guide RNA and Cas12b were 250 pM), 1 pL of a target DNA or 1 pL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1) (with a final concentration of 500 nM), 2 pL of a NEB 10X 3.1 buffer and 0.5 pL of an RNase inhibitor (40 U / pL). After mixing homogeneously, the reaction was conducted at 48 °C for 30 minutes. After that, it was inactivated by heating over a flame at 98 °C for 5 minutes in a PCR instrument.

[0246] 4. Detection of Cas12b transcleavage activity by a fluorescence microplate reader method:

[0247] 20 pL of the inactivated reaction solution were 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. Characteristics of cis-cleavage of Cas12a in the cleavage of a target single-stranded DNA:

[0248] First, to test the single-stranded DNA cleavage characteristics of Cas12a, several crRNAs targeting a short single-stranded DNA (DNMT1-3) (Table 1) are designed, labeled with 5(6)-carboxyfluorescein (FAM) at the 3' end. After cleavage by FnCas12a, the reaction product is analyzed by denatured urea-polyacrylamide gel electrophoresis (ureaPAGE). It is found that single-stranded DNA cleavage by Cas12a is programmed. That is, the cleavage site is near the 22nd base (21aa 23abases) of the target sequence counting from the 3' end base to the 5' end of the first target sequence paired with the crRNA guide sequence, as shown in FIGURES 1A and 1C. Cleavage of double-stranded DNA by Cas12a requires a PAM sequence, while cleavage of single-stranded DNA by Petition 870200036677, dated 03 / 19 / 2020, p. 46 / 85 40 / 60 Cas12a does not require the PAM sequence (FIGURES 1A, 1B, and 2), which is similar to Cas9-mediated single-stranded DNA cleavage. However, Cas12a-mediated single-stranded DNA cleavage activity depends on a stem-loop structure in the crRNA, as shown in FIGURE 1A, while Cas9 still shows weak cleavage activity for single-stranded DNA with only a 20 nt complementary RNA sequence. The stem-loop structure of the crRNA is important for stabilizing the Cas12a structure, which is why the crRNA ring structure is necessary for Cas12a-mediated single-stranded DNA cleavage. It is further tested whether a shorter lead-sequence crRNA can pass through the Cas12a single-stranded DNA cleavage site in such a way that the cleavage is outside the recognition site.When the guide sequence length is 16 nt, 18 nt, and 20 nt, all these crRNAs lead to cleavage by Cpf1 near base 22, as shown in FIGURES 1B and 1D, meaning that the cleavage site is 4 nt, 2 nt, or 0 nt outside the recognition site. Next, the efficiency of Cas12a cleavage on different substrates is tested using substrates of double-stranded DNA and single-stranded DNA, respectively, as shown in FIGURE 1F. Similar to the Cas9 cleavage situation, the cleavage of single-stranded DNA is slower than that of double-stranded DNA, as shown in FIGURES 1E to 1G. These results indicate that the recognition and cleavage mechanism of single-stranded DNA by Cas12a may be different from that of double-stranded DNA, which is a low-efficiency recognition and cleavage method independent of PAM; and the PAM sequence accelerates the recognition and / or division of the target double-stranded DNA by Cas12a. Transcleavage characteristics of Cas12a in the cleavage of single-stranded DNA:

[0249] When the target single-stranded DNA is labeled at the 3' end, Cas12a cleaves near the 22a base, as shown in FIGURE 1. However, when it is labeled at the 5' end, no cleavage product band of the predicted size is observed, but a short (<6 nt) FAM-labeled product is produced, as shown in FIGURE 3B. Through detailed experiments, once the ternary complex Cas12a / crRNA / target single-stranded DNA is formed, the target single-stranded DNA (DNMT1-3) (Table 1) labeled at the 5' end is cleaved and a labeled product is produced. Petition 870200036677, dated 03 / 19 / 2020, page 47 / 85 41 / 60 with FAM is produced, as shown in FIGURE 3C. Furthermore, the ternary complex also cleaves single-stranded DNA that does not have a sequence complementary to the crRNA (i.e., collateral single-stranded 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 the target single-stranded DNA is labeled at the 3' end, 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 / target single-stranded DNA, and the target single-stranded DNA is protected so that its labeled 3' end is shielded from exposure to a nuclease active site of the ternary complex. These cleavage processes can be as shown in FIGURE 3A.

[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 on plasmid DNA (as shown in FIGURE 4B), and all ternary Cas12a complexes exhibit cis and trans cleavage activities on single-stranded DNA (as shown in FIGURES 4C and 4D). This shows that cis and trans Cas12a activity on single-stranded DNA is a common phenomenon. The key cis and trans sites and mechanism for cleaving single-stranded DNA by Cas12a.

[0251] To determine the key amino acid residues related to cis and trans activities in single-stranded DNA in Cas12a, several candidate Cas12a residues are mutated to perform the activity test. First, three single amino acid mutants of FnCas12a (H843A, K852A, and K869A) are purified and tested, and their residues are related to RNase activity. The results of the trans activity study 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] Next, when the endonuclease active sites in FnCas12a, namely the RuvC domain (D917A, E1006A or D1255A) and Nuc domain (R1218A) sites are mutated, the two cis and trans cleavage activities of this Cas12a Petition 870200036677, dated 03 / 19 / 2020, page 48 / 85 42 / 60 mutated single-stranded DNA are affected, as shown in FIGURES 5B and 5D. These results indicate that the key Cas12a site for cleaving a target double-stranded DNA is closely related to cis and trans cleavage activities in single-stranded DNA.

[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 cavity, as shown in FIGURES 6A and 6B. When comparing the endonuclease catalytic residues of Cas12b (i.e., C2c1) and Cas12a, it is most likely that these sites play similar roles in the cleavage and functions of Cas12b (i.e., C2c1) and Cas12a. The results of an in vitro single amino acid mutation experiment show that it is consistent with the above hypothesis. That is, Cas12a likely cleaves two strands through only one RuvC catalytic cavity.

[0254] Transcleavage activity of a Cas12a complex: In the structure of a Cas12b complex (i.e., C2c1) with an 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 collateral single-stranded DNA substrate in Cas12a. Combined with a single amino acid mutation experiment, it is proposed that target DNA, non-target DNA, and collateral single-stranded DNA are all cleaved in the single RuvC cavity in Cas12a, as shown in FIGURES 6D, 6E, and 6F. The ternary Cas12a complex has collateral single-stranded DNA transcleavage activity, while the reason why a monomer or binary complex does not possess collateral single-stranded DNA transcleavage activity can be explained by comparing the structures of the monomer, binary, and ternary complexes.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 / target DNA complex is converted into a double-sheet structure, thus exposing the catalytic cavity to perform transcleavage of collateral single-stranded DNA (as shown in FIGURE 6H). Establishing a method for nucleic acid detection.

[0255] Based on the characteristics of Cas12a, a method was developed for the specific detection of nucleic acid molecules, called Petition 870200036677, dated 03 / 19 / 2020, page 49 / 85 43 / 60 HOLMES (a one-hour, low-cost, multipurpose, efficient, simple test). Just like the technology's name suggests, it was characterized by a simple testing method that lasts one hour and is low-cost, multi-purpose, and highly efficient.

[0256] In the entire reaction system, the method can be divided into two main steps, one is the amplification of a template 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 nucleic acid detection in the second step, such as the RPA isothermal amplification method, etc. The initial nucleic acid is not limited to double-stranded DNA, but can also be single-stranded DNA; or even RNA can still be detected after reverse transcription, therefore this method is suitable for various types of nucleic acid molecules. For the nucleic acid detection phase, three components are key to the experiment, namely, Cas12a, crRNA, and a nucleic acid probe.In addition to the 10 Cas12as mentioned in the example (these 10 proteins are randomly selected), other Cas12a proteins are also suitable for this method. Furthermore, other types of Cas proteins (e.g., a C2c1 protein) are also suitable for the claimed scope of the present invention: as shown by experimental results, Alicyclobacillus acidoterrestris Cas12b (i.e., C2c1) also possesses co-lateral single-stranded DNA transcleavage activity similar to that of Cas12a, and its complex with target crRNA / DNA can also cleave co-lateral single-stranded DNA.

[0257] For the guide crRNA, it will be more stable in the system after being engineered, for example, by being manually modified. 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, provided that the nucleic acid probe is cleaved to produce detectable differences. Alternatively, the nucleic acid probe can also be engineered to fluoresce upon binding to a compound in order to detect whether the probe is cleaved.

[0258] Furthermore, it must be understood that, after reading the teachings Petition 870200036677, dated 03 / 19 / 2020, pp. 50 / 85 44 / 60 above the present invention, those skilled in the art may make various alterations or modifications to the present invention, and such equivalent forms also fall within the scope defined by the claims appended to this application. Table 1 Characteristic divagem substrate sequences related to the Cas12a experiment _ Name Oliqo Sequence (5'-3') SEQ ID NO.: DNMT 1-3-Falvo aatgtttcctgatggtccatgtctgttactcgc ctgtcaagtggcgtgac I ' DNMT 1-3-R alvo gtcacgecacttgacaggcgagtaacagacatg caccat caccat 2 DNMT 1-3-R-FAM-3' alvo gtcacgccacttgacaggcgagtaacagacatg gaceatcaggaaacatt-FAM 3 DNMT 1-3-R-FAM-5' alvo FAM-gtcacgccacttgacaggcgagtaacaga catggaccatcaggaaacatt T1-4_Falvo tttctgtttgttatcgcaactttctactgaatt caagctttactctagaaagaggagaaaggatcc 5 _ T1-R alvo ggatccttctctctctcttagtaaagcttg aattcagtagaaagttgcgataacaaacagaaaa 6 ' T1-F-FAMalvo-FAM-ttctctctctctctc aattcaagctttactctagaaagaggagaaagg atcc 7 T1-R-FAM alvo ggatccttctctctctcttagagtaaagcttg aattcagtagaaagttgcgataaaaacagaaaa -FAM 8 T1-FAM-3' -F alvo tttctgtttgtgtcgcaactctactc caagctttactctagaaagaggagaaaaacaaaca IO Petition 870200036677, dated 03 / 19 / 2020, p. 51 / 85 45 / 60 gaaa DNMT1-3-RI I I- FAM-3' alvo gtcacgccacttgacaggcgagtaacagacatg gaccatcaggTTTcatt-FAM 11 DNMT1-3-R-CCC- FAM-3' alvo gtcacgccacttgacaggcgagtaacagacatg caccat-FACCTC2 DNMT1-3-R-GGG- FAM-3' alvo gtcacgccacttgacaggcgagtaacagacatg gaccatcaggGGGcatt-FAM 13 DNMT1-3-F-AAA alvo aatgAAAcctgatggtccatgtctgttactcgc ctgtcaagtggcgtc DNMT1-GG13-GG13-GF-GF-FAM-FAM aatgGGGcctgatggtccatgtctgttactcgc ctgtcaagtggcgtgac 15 DNMT1-3-F-CCC , alvo aatgCCCcctgatggtccatgtctgttactcgc ctgtcaagtggcgtgac 16 T1-1-Raivo acaaacagaaa 17 cRaa algaa ga-aa 18 T1-12-Ralvo aagttgcgataaaaacagaaa 19 , T1-18-Ralvo agtagaaagttgcgataaaaaaaaaaa 20 T1-24-Ralvo gaattcagtagaaagttgcgataaaaaaaaa a 21-R24-alvosomente gaattcagtagaaagttgcgataa 22 T1 -18-somente-R alvo agtagaaagttgcgataa 23 T1-12-somente-R alvo aagttgcgataa 24 T1 -6-somente-R alvo cgataa 25 N25-5' FAM FAM-NN-NN3NNNNNNNN NNN2NNNNNN WOMAN NNNNNNNNNNNNNNNNNNNNNNN-WOMAN 27 Table 2. Names and Gl numbers of the Cas12a and Cas12b proteins (i.e., C2c1) Petition 870200036677, dated 03 / 19 / 2020, p. 52 / 85 46 / 60 Name Number Gl Species FnCasl2a 489130501 Francisella tularensis AsCas12a 545612232 Acidaminococcus sp. BV3L6 LbCasl2a 917059416 Lachnospiraceae bacterium ND2006 Lb5Casl2a 652820612 Lachnospiraceae bacterium NC2008 HkCasl2a 491540987 Helcococcus kunzii ATCC 51366 0sCasl2a 909652572 Oribacterium sp, NK2B42 TsCasl2a 972924080 Thiomicrospira sp. ΧΞ5 BbCasl2a 987324269 Bacteroidales bacterium ΚΛ00251 BoCas12a 496509559 Bacteroidetes oral taxon 274 str. F0058 Lb4Casl2a 769130406 Lachnospiraceae bacterium MC2017 C2cl 1076761101 Alicyclobacillus acidoterrestris Table 3 Information on plasmids Petition 870200036677, dated 03 / 19 / 2020, p. 53 / 85 47 / 60 Plasmids or Strains Relevant properties or genotypes Sources Plasmids pET28a-TEV pET28a with the thrombin cleavage site modified to the ! site TEV protease cleavage (Carneiro, Silva et al. 2006) pET28a-TEV-F nCas12a pET28a-TEV carrying FnCas12a (Li, Zhao et al. 2016) pET28a-TEV-AsCas12a pET28a-TEV carrying AsCas12a (Li, Zhao et al. 2016) pET28a-TEV-LhCas12a pET28a-TEV carrying LbCas12a (Lei, Li et al. 2017) pET28a-TEV-Lb5Cas!2a pET28a-TEV carrying Lb5Cas12a The present invention pET28a-TEV-HkCas12a pET28a-TEV carrying HkCas12a The present invention pET28a-TEV-0sCas 12a pET28a-TEV carrying OsCas12a The present invention pET28a-TEV-TsCasl2a pET28a-TEV carrying TsCas12a The present invention pET28a-TEV-BbCasl2a pET28a-TEV carrying BbCas12a The present invention pET28a-TEV-BoCasl2a pET28a-TEV carrying BoCas12a The present invention pET28a-TEV-Lh4Cas!2a pET28a-TEV carrying Lb4Cas12a The present invention Petition 870200036677, dated 03 / 19 / 2020, pp. 54 / 85 48 / 60 PET2Ka-TEV-FnCas12a-K869 A pET28a-TEV carrying FnCas12a-K869A The present invention pET28a-TEV-FnCas12a-K852 A pET28a-TEV carrying FnCas12a-K852A The present invention pET28a-TEV-FnCas12a-H843 A pET28a-TEV carrying FnCas12a-H843A The present invention pET28a-TEV-FnCas12a-R1218A pET28a-TEV carrying FnCas12a-R1218A The present invention pET28a-TEV-FnCaEl2a-E100EA pET28a-TEV carrying FnCas12a-E1006A The present invention pET28a-TEV-FnCasl2a—D917 A pET28a-TEV carrying FnCas12a-D917A The present invention pET28a-TEV-FnCasl2a-Dl25 5Λ pET28a-TEV carrying FnCas12a-D1255A The present invention pET28a-TEV-C2c1 pET28a-TEV carrying C2c1 The present invention Table 4 Primers used in the HOLMES method test Name Oligo Sequence (5'-3') SEQIDNO.: T1-R alvo ggatccttctctcctctctctagagtaaagcttgaa ttcagtagaaagt tgcgataaaaaacagaaa 28 M13F-47 cacaattccacacaacatacgagccgga 29 M1-Rcct38Gccagcagcagcagcag 30 T1-F alvo agttttgttatcgcaactttctactgaattc 31 T1-F-1Aalvo agttttgAtatcgcaactttctactgaattc 32 T1-F-2Aalvo agttttgtAatcgcaactttctactgaattc 33 T1-F-3Tal agttttgttTtcgcaactttctactgaattc 34 T1-F-4Aalvo agUttgttaAcgcaacr. ttctactgaattc 35 T1-F-5G alvo agttttgttatGgcaact t tctactgaattc 36 T1-F-6C alvo agttttgttatcCcaactttctactgaattc 37 T1-F-7G alvo agttttgttatcgGaactttctactgaattc 38 Petition 870200036677, of 19 / 03 / 2020, p. 55 / 85 49 / 60 T1-F-8Talvo agttttgttatcgcTactttctactgaattc 39 T1-F-9Talvo agttttgttatcgcaTctttctactgaattc 40 T1-F-10G alvo agttttgttatcgcaaGtttctactgaattc 41 T1-AAAN-F alvo aaaagttatcgcaactttctactgaattc 42 T1-F-11Aalvo agttttgttatcgcaacAttctactgaattcggtc atag 43 T1-F-12Aalvo agttttgttatcgcaactAtctactgaattcggtc atag 44 T1-F-13Aalvo agttttgttatcgcaacttActactgaattcggtc atag 45 T1-F-14G alvo agttttgttatcgcaactttGtactgaattcggtc atag 46 T1-F-15A alvo agttttgttatcgcaactttcAactgaatteggtc atag 47 T1-F-16Talvo agttttgttatcgcaactttctTctgaattcggtc atag 48 T1-F-17G alvo agttttgttatcgcaactttctaGtgaattcggtc atag 49 T1-F-18Aalvo agttttgttatcgcaactttctacAgaatteggtc atag 50 T1-PAM1A-F alvo agtttAgttatcgcaactttctactgaattc 51 T1-PAM2A-F alvo agttAtgttatcgcaactttctactgaattc 52 T1-PAM3A-F alvo agtAttgttatcgcaactttctactgaattc 53 gyrB-F AGTTGTCGTTCCTCAACTCCGGCGTTTC 54 gyrB-R TCGACGCCAATACCGTCTTTTTCAGTGG 55 1-5082-F CTGCCTTTGCTTCTACCTTTGCCTGT 56 1-5082-FT TTGCTTCTACCTTTGCCTGTTCTGG 57 1-5082-R TTTTCTGGCTGGGGATGGCCGATGG 582-r$1467558-F AGCAATAACACTAATATTGATTCCTTCAGATATGG ACTCCTTTCATAGTA 59 Petition 870200036677, dated 03 / 19 / 2020, pp. 56 / 85 50 / 60 2-rsl467558-F-T TTGATTCCTTCAGATATGGACTCCTTTCATAGTAT AACG 60 2-rsl467558-R TGAGCATCGTTATTCTTACGCGTTGTCATTGAAAG AG 61 3-rs2952768-F AGCCTGGGCAACGAGTGAAACTCTG 62 3-rs2952768-R ACAGGAGGGACAAAGGCCTAAGTGTCC 63 3-rs2952768-R-C CATCATAGGATTGGGAAAAGGACATTTCAGTCATT CAG 64 4-rs4363657-F AGAGTCCTTCTTTCTCAATTTTTCAGAATAATTTA GTACTTTGGGTAC 65 4-r$4363657-lí CAGTACTGAAAAAACCTGCCTATCAATAAAAGCCC TAGAC 66 5-rs601338-F GCTTCACCGGCTACCTTTGCTCCT 67 5-rs60l338-R TTCACCTGCAGGCCCCGCAGG 68 34-TP53-T24-F CCTGACTTTCAACTCTGTCTCCTTCCTCTTTTTAC AGTA 69 34-TP53-T24-R TGCTGTGACTGCTTGTAGATGGCCATGG 70 41-rsl014290-F AGTTTCCAGACCTCAGTGCACAAGATACTTTTCTA C 71 41-rsl()14290-F-G ACCTCAGTGCACAAGATACTTTTCTACGTCATCCA C 72 41-rsl014290-R AGCTCCAGTGGATGGAAGATCTTTGAGATCCAG 73 42-rs6449213-F AGTCAAAGAGATTCATGCCTGGGACTTTAATCACA TTTAT 74 42-rs64492l3-F-C ATGCCTGGGACTTTAATCACATTTATCGGAAGG 75 42-rs6449213-R CAAATCTGTCTCCACCTCTCAGCTCACCTTG 76 43-rs737267-F TTCTTGAACCCAAACTCACCTGGCATTTAAACTG 77 43-rs737267-F-AAAACTCACCTGGCATTTAAACTGACTCTGTAAG 78 43-rs737267-FT AAACTCACCTGGCATTTAAACTGTCTCTGTAAG 79 43-rs737267-R TGCCGAGGCTGAGTTCAGCTACTCTCC 80 Petition 870200036677, dated 03 / 19 / 2020, p. 57 / 85 51 / 60 44-rsl260326-F ACACAGCACCGTGGGTCAGACCTTGC 81 44-r$1260326-FC TGGGTCAGACTTTGCCGGTGAGAGTC 82 44-rsl260326-FT TGGGTCAGACTTTGCTGGTGAGAGTC 83 44-rsl260326-R AGCAGTGGCCATGTGATGCTGATGATG 84 45-rs642803-F CCCCGGCTCTGTTGGCTTTGAGAATTG 85 45-rs642803-FC CTCTGTTGGCTTTGAGAATTGCCTGTCTGTGTC 86 45-rs642803-FT CTCTGTTGGCTTTGAGAATTGTCTGTCTGTGTC 87 45-rs642803-R ACCGATACCTGGCAGCCCTTGGATG 88 EIEX-N12-BHQ1 |[EX-NNNNNNNNNNNN-BHQ1 89 Table 5 Template sequences for crRNA transcription Nome Oligo Sequence (5'-3') SEQID NO.: T7-crRNA-F GAAATTAACGACTCACTATAGGG 90 T7-T1-24-R gattcagtagaagttgcgataaATCTTACAACAGTAGA AATTCCCTATAGTGAGTCGTATT-7 TAATTTC-91 agaaagttgcgataaATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 92 T7-T1-16-R tagaag 1t gcga taATCTACACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 93 T7—T1 — 17—TCAAttgACCCAA gragaaag TATAGTGAGTCGTATTAATTTC 94 T7-T1-18-R ag ta gaa agttgcgataaAT CTACAACAGTACAAAΊ CC CTATAGTGAGTCGTAT TAATTTC 95 T7-crRNA-[)NM T-23nt-R GAGTAACAGAGAGATGATCAACCATCA ATTCCCTATAGTGAGTCGTATTAATTTC 96 T7-crRNA-l)NM T-(-8)-R gabatggaccatcaggaaacattATCTACAACAGAAA ATTCCCI ATAGTGAGTCGTATTAATTTC 97 T7-crRNA-[)NM T-(+4)-taaAGCTIAA AgacaCACAR ATTCCCTATAGTGAGTCGTATTAATTTC 98 T7-crRNA-DNM tgacaggcgagtaacagacatggATCTACAACAGTAGAA 99 Petition 870200036677, dated 19 / 03 / 2020, p. 58 / 85 52 / 60 τ- (+8) -R ATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-DNM T-l6nt-R agacatggaccatcagATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC ICO T7-crRNA-ÜNM T-18nt-R acagacatggaccatGACCATTACCATTACCATTACCATTACCATTC CTATAGTGAGTCGTATTAATTTC 101 T7-crRNA-DNM T-20nt-R taacagacatggaccatcagATCTACAACAGTAGAAATT CCCTATAGTGAGTCGTATTAATTTC 102 T7-DNMT-(-8)sem loop-R gacatggaecatcaggaacattCC CTATAGATTTC0GTAATT33 T7-DNMT(+4)-sem loopR aggcgagtaacagacatggaccaCCCTATAGTGAGTCGT ATTAATTTC 104 T7-DNMT(+8)-sem loopR.LgacaggcgagtaacagacatggCCCTATAGTGAGTCGT ATTAATTTC 105 T7-crRNA-rs50 82-T CCTCTTCCCAGAACAGGATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 106 T7-crRNA-rs50 92G CCCACCACCAGTCGATTGATTGATTGATC ATAGTGAGTCGTATTAATTTC 107 T7-crRNA- rs 1467558-T CTGAAGCGTTATACTATATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 108 T7-crRNA- rs!467558-C CTGAAGCGTTGTACTACTATCTACCTATCTACCTATTATTTTC ATAGTGAGTCGTATTAATTTC 109 T7-crRNA-rs29 52768-Γ-16ηΐ TTTTATCTGAATGATTATCTACAACAGTAAATTCCCTA TAGTGAGTCGTATTAATTTC 110 T7-crRNA-rs29 52768-C-16tn TTTTATCTGAATGACTATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 111 T7-crlíNA- rs4363657-T AAAAAGAGTGAGTACCATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 1 12 T7-crRNA-43636-CRNA AAAAAAGAGTGGGTACCATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 113 T7-crRNA-rs60 GGTAGAAGGTCCAGGAGATCTACAACAGTAGAAATTCCCT 114. Petition 870200036677, of 19 / 03 / 2020, p. 59 / 85 53 / 60 1338-G ATAGTGAGTCGTATTAATTTC T7-crRNA-rs60 1338-A GGTAGAAGGTCTAGGAGATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 115 T7-erRNA-34-T >53-T24-C-16n t GGGCAGGGGAGTACTGATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 116 T7-crRNA-34-T >53-T24-G-16n t GGGCAGGGGACTACTGATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 117 T7-crRNA-41-r s1014290-A-15 nt TCAGTGGATGATGTAATCTACAACAGTAGAAATTCCCTAT AGTGAGTCGTATTAATTTC 118 T7-crRNA-41-r 81014290-6-15 nt TCAGTGGATGACGTAATCTACAACAGTAGAAATTCCCTAT AGTGAGTCGTATTAATTTC l 19 T7-crRNA-42-r S6449213-C GGAAATTCTCCTTCCGAATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 120 T7-crRNA-42-r S6449213-T GGAAATTCTCCTTCCAAATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 121 T7-crRNA-43-r s737267-A-16n t TCTTACAGAGTCAGTTATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 122 T7-crRNA-43-r s737267-G-16n t TCTTACAGAGCCAGTTATCTACAACAGTAGAAATTCCCTA TAGTGAGTCGTATTAATTTC 123 T7-crRNA-43-r s737267-T GTCTTACAGAGACAGTTATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 124 T7-crRNA-44-r S1260326-C-15 ntCTGGACTCTCACCGGATCTACAACAGTAGAAATTCCCTAT AGTGAGTCGTATTAATTTC 125 Petition 870200036677, dated 03 / 19 / 2020, pp. 60 / 85 54 / 60 P7-erRNA-44-r S1260326-T-15 □ t CTGGACTCTCACCAGATCTACAACAGTAGAAATTCCCTAT AGTGAGTCGTATTAATTTC 126 T7-crRNA-45-r s642803-C CACAGACAGGCAATTCTATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 127 T7-crRNA-45-r 5642803-T CACACAGACAGACAATTCTATCTACAACAGTAGAAATTCCCT ATAGTGAGTCGTATTAATTTC 128 T7-crRNA- gyrB TCGCGCTTGTCGCGCAGACGAATGATCTACAACAGTAGAA ATTCCCTATAGTGAGTCGTATTAATTTC 129

[0259] Primers used for detection by DNA amplification via LAMP combined with Cas12a: Table 6 Primers used to amplify gyrB-1 Nome Sequência SEQ ID NO.: LAMP-gyrB-l-F3 CATGGTGCGTTTCTGGCC 13Ü LAMP-gyrB-l-IJ3 CGGCGTTTTGTTCTTGTTCA 131 LAMP-gyrB-l-FTP ACAACTCACGCAGACGTTTCGCAACCTTCA CCAATGTGACCG 132 LAMP-gyrB-l-BlP GTTCCTCAACTCCGGCGTTTCGATGCCGC CTTCATAGTGG 133 LAMP-gyrB-l-LoopF ​​CAGAATTTCATATTCGAACT 134 LAMP-gyrB-l-LoopB GACGGCAAAGAAGACCACTT 135 Tabela 7 Primers used to amplify gyrB-2 Petition: 870200036677, on 03 / 19 / 2020, page. 61 / 85 55 / 60 Nome Sequência SEQIDNO.: LAMP-gyrB-2-F3 CGACGGCAAAGAAGACCA 136 LAMP-gyrP-2-lí3 AGCCTGCCAGGTGAGTAC 137 LAMP-gyrB-2-FIP cgggtggatcggcgttttgttcactatgaa GGCGGCATCA 138 LAMP-gyrB-2-BTP GTATTGGCGTCGAAGTGGCGTTCGCTGCGG AATGTTGTTG 139 LAMP-gyrP-2-LoopF ​​TTGTTCAGA'I ATTCAACGAACG 140 LAMP-gyrB-2-LoopB GTGGAACGATGGCTTCCAGG 141 Tabela 8 primers used to amplify at location rs1467558 Name Sequence SEQIDNO.: LAMP-rs 1467558-1'3 CAGCTGTAGACCATAAGCC 142 LAMF-rsl467558-B3 GTGGCTGAGCATCGTTAT 143 LAMP-rsl467558-FI P ACTATGAAAGGAGTCCATATCTGAAGGAAT TCAGGTAGTGGTTTGGGA 144 LAMP-rsl467558-Bl P GCTTCAGUCTACrGCAAATCCTACGCGTTG TCATTGAAAG 145 LAMP-rsl467558-Lo opl·' TCAAíATTAGTGFrATTGCTTG 146 LAMP-rsl467558-Lo opB TGGTGGAAGATTTGGACAGGAC 147 Table 9 Primers used to amplify an rs5082 site Petition 870200036677, dated 03 / 19 / 2020, pp. 62-85 56 / 60 Name Sequence SEQ ID NO.: LAMP-rs50«2-F3 GCTGGAAAGGTCAAGGGAC 14« LAMP-rs5082-33 GGGGTTrGTTGCACAGTCC 149 LAMP-rs5082-FlP CAAAGGTAGAAGCAAAGGCAGGAGGTTTGC CCAAGGTCACACAG 130 LAMP-rs5082-BTP CTGGGAAGAGGGAGGGCTCAGTGTTGCCAC ACTTTCAUTGG 151 LAMP-rs5082-LoopF ​​​​GTGAGUGGGTGGGGTGCT 152 LAMP-rs5082-LoopB TCTAAG KTTCCAGCACGGGATG 153 Table 10 Primers used for detection by amplification of RPA combined with Cas12 Name Sequence SEQ ID NO.: RPA-gyrB-lF ATATGAAATTCTGGCGAAACGTCTGCGTGAGTTG 154 RPA-gyrB-2-F AAACGTCTGCGTGAGTTGTCGTTCCTCAACTCC 155 RPA-gyrB-R ACTTCGACGCCAATACCGTCTTTTTCAGT 156 Table 11 Primers used to determine Cas12b with transcleavage activity: Petition 870200036677, dated 03 / 19 / 2020, pp. 63 / 85 57 / 60 Name Oligo Sequence (5'-3') SEQIDNO.: pUC18-lF ATCTGAGAAGTGGCACTTÁFGGCAACTTTCTACTGAGGTC ATAGCTGTTTCCTGTGTGA 157 pLCl8-1-R GTCCTCIAGACCCCIATAGTGAGT^ GATTACGAATTCGGGTCG15 CCACTTTCCAGG1GGCAAAGCCCGTTGAGCTTCTCAAA TCTGAGAAGTGGCACTTATC 159 PUC18-2-R TGGAAAGTGGCCATTGGCACACCCGTTGAAAAATTCTG TCCTCTAGACUCCTATAGTGA 160 T7-crRNA-F GAAATTAATAGGACTATAGCACTATA11 ZL-sgRNA-Tl-R rCAGTAGAAAGTTGCGAFAAGTGC 162 ZLsgRNA-DNMTl- 3-R AACAGACATGGACCATCAGGGTG 163 T1-F alvo TTICTGTTTGTTATCGCAACTTTCTACTGAATTCAAGC TTTACTCTGAAGAGGAGGAGGA164 or GGATCCTTTCKCTCTHCT AGIAGAAAGTTGCGATAACAAACAGAAA 165 DNMT1-3-R- FAM-5' alvo GTCACGCCACTTGACAGGCGAGTAACAGACATGGACCA TCAGGAAACATT 166 T1-R alvo GGATCCTTTC'IOCTCT'n ACTAACTAACTAACTAAGAGATTAAGATTAAGATT167 1 T1-Falvo TTTCTGTTTGTTATCGCAACTTTCTACTGAATTCAAGC 'F'rTACTCTAGAAAGAGGAGAAAGGA'FCC 168 Table 12 Primers used for Cas12b trans reaction sensitivity testing Petition 870200036677, of 19 / 03 / 2020, p. 64 / 85 58 / 60 Oligo Name Sequence (5'-3') SEQID NO.: sgfí\A-0hm-3- CCTGATGGTCCATGTCTGTTGGTCATAGCTGTTTCCTGTG TG 169 sgRXA-DNMTl-3- R TGGACCATCAGGGTGCCACTTCTCAGATTTGAG 170 T7-crRNA-F GAAATTAATACGACTCACTATAGGG 171 ZLsgRNA-DNMTl- 3-R AACAGACATGGACCATCAGGGTG 172 DNMT1-3(TTC PAM)-F AATGTTCCCTGATGGTCCATGTCTGTTACTCGCCTGTC AAGTGGCGTGAC 173 DNMT1-3(TTC PAM)-R GTCACGCCACTTGACAGGCGAGTAACAGACATGGACCA TCAGGGAACATT 174 LAMP-DNM-E3 gtgaacgttcccttagcact 175 lamp-dnm-b:? gggagggcagaac tagtcc 176 LAMP-DNM-FIP cgccacttgacaggcgagtaac tgccacttattgggtc age 177 LAMP-DNM-BTP gcgtgttccccagagtgacttagcagcttcctcctcct t 178 LAMP-DNM-LoopF ​​aggaaacattaacgtaetgatg 179 EAMP-DW-LoopB ttccttttatttcccttcagc 180 DNMT1-3(TTC PAM)-R GTCACGCCACTTGACAGGCGAGTAACAGACATGGACCA TCAGGGAACATT 181 DNMT1-3(TTC PAM)-F AATGTTCCCTGArGGTCCATGTCTGTTACTCGCCIGTC AAGTGGCGTGAC 182 Table 13 Other sequences involved in the present invention Petition 870200036677, dated 03 / 19 / 2020, pp. 65 / 85 59 / 60 Name Sequence SEQ ID NO.: sgRNA sequence AacCas12b GTCTAGAGGACAGAATTTTTCAACGGGTGTGCCAATGG CCACTTTCCAGGTGGCAAAGCCCGTTGAGCITCTCAAA TCrGAGAAGTGGCACcctgatggtccatgtctgtt 183 Guide sequence directed to a DNMT-1-3 target cctgatggtccatgtctgtt 184 Single-stranded target sequence gtcacgccacttgacaggcgagtaacagacatggacca tcagggaacatt 186 Double-stranded target sequence: gtcacgccacttgacaggcgagtaacagacatggacca tcagggaacatt 186 Amino acid sequence of the AacCas12b protein W'KSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTE WLSLLRQENlARRSPNGDGEQECDKTAEECKAELLERb RARÜVENGI IRGPAGSDDELLQLARQLYELl ΛΊΌΑIGAK GDAQQ l ARKFLSPLADKDAVGGLGIAKAGNKPRWVRMR EAGEPGWEEEKEKAlirRKSADRTADVLliAl.ADFGLKPl. ilRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERM MSWESWXQRVGQEYAKIATQKNRFEQKNFVGQEHLVIIL VNQLQyDMKEASPGLESKEQTAHYVTGRAI.RGSDKVFE KWGKLAPDAPFDLYDAElKNVyRRNTRRFGSHDLFAKL AEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATF TLPDATAIIPIWTRFDKLGGNLHQYTFLFNEFGERRIIAI RFHKLLKVENGVARDDEPSEVPNPQLPN 1ALYFRIÍYGAEQIIFTGEFGGAKIQCRRÜQLAHMHRR rííardvylnvsvrvqsqsearrppyaavfrlvgdn HRAFVHFDKbSDYLAEnPDDGKLGSEGLLSGLRVMSVr) ULIJnSASISVFimRKOELKRNKGRNKGVGVNI)7KGVNI. Petition 870200036677, of 19 / 03 / 2020, p. 66 / 85 60 / 60 NLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLR ​​TQLAYLRLLVRCGSEDVGRRERSWAKLIEQP VDA ANIIM TPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRV WRIIMGKQVRDWRKDVRSGERPK[RGYAKDVGGNSIQ IEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLR Ell 1 D[IAKEDRLKKLADR [ IMEALGYVYALDERGKGKWV AKYPPCQLILLEELSEYQFNNDRPPSENNQbMQWSHRG VFQELINQAQVHDLLVGSSRFDYAFGARTGARTG RRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADD LIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRL WSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYS NKVFYTNTGVTYERERGKKRKVEKVEQEELL VEADEAREXVLMRDPSGIINRGNWTRQKEFWSMVNQ RIEGYLVKQ1RSRVPLQDSACENTGD1 *

[0260] All documents mentioned in the present invention are incorporated herein 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 may make various alterations or modifications to the present invention, and such equivalent forms also fall within the scope defined by the claims appended to the present application. Petition 870200036677, dated 03 / 19 / 2020, pages 67 / 85

Claims

1 / 4 CLAIMS 1. A method for detecting target nucleic acid molecules comprising a target sequence, characterized in that it comprises the steps of: I) adding (a) a guide RNA comprising a target site that specifically hybridizes with the target sequence, (b) a Cas12 protein having collateral cleavage activity, wherein the collateral cleavage activity of the Cas12 protein is activated when specific hybridization of the guide RNA and the target sequence occurs, (c) a detectably labeled single-stranded DNA probe susceptible to the collateral cleavage activity of the Cas12 protein, and (d) a buffer solution in a system containing the target nucleic acid molecules to be detected; and II) then detecting cleavage of the nucleic acid probe.

2. Method according to claim 1, characterized in that the Cas12 protein is Cas12a or a Cas12 protein that has a collateral single-stranded DNA cleavage activity similar to that of Cas12a.

3. Method according to claim 1, characterized in that the single-stranded DNA probe is 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.

4. Method according to claim 1, characterized in that the Cas12 protein is Cas12b.

5. Use of a Cas12 protein in a method for detecting a target nucleic acid molecule comprising a target sequence, characterized in that when a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex cleaves non-target single-stranded DNA molecules in the system; wherein the guide RNA comprises a target site that hybridizes specifically with the target sequence; wherein the Cas12 protein has collateral cleavage activity, wherein the collateral cleavage activity of the Cas12 protein is activated when specific hybridization of a guide RNA and the target sequence occurs; Petition 870260076986, dated 07 / 31 / 2026, p. 11 / 19 2 / 4 wherein the non-target single-stranded DNA molecule is a detectably labeled single-stranded DNA probe susceptible to the collateral cleavage activity of the Cas12 protein.

6. Detection system for detecting a target nucleic acid molecule comprising a target sequence, characterized in that it comprises: (a) a Cas12 protein having collateral cleavage activity, which is Cas12a or a Cas12 protein with collateral single-stranded DNA cleavage activity similar to that of Cas12a; (b) a guide RNA that guides the Cas12 protein to bind specifically to the target nucleic acid molecule; and comprises a target site that hybridizes specifically with the target sequence; wherein the collateral cleavage activity of the Cas12 protein is activated when specific hybridization of the guide RNA and the target sequence occurs; (c) a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity of the Cas12 protein, which is single-stranded DNA; and (d) target nucleic acid molecules comprising a target sequence; and (e) a buffer solution in which the target nucleic acid molecule is target DNA.

7. Detection system, according to claim 6, characterized in that the Cas protein that has collateral single-stranded DNA cleavage activity similar to that of Cas12a is Cas12b.

8. Kit for detecting a target nucleic acid molecule comprising a target sequence, characterized in that it comprises: i) a first container and a Cas12 protein having collateral cleavage activity in the first container, the Cas12 protein being Cas12a or the Cas12 protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a; ii) a second container and a guide RNA in the second container, the guide RNA guiding the Cas12 protein to bind specifically to the target nucleic acid molecule, and comprising a target site that hybridizes specifically with the target sequence; Petition 870260076986, dated 07 / 31 / 2026, p.12 / 19 3 / 4 wherein the side-cleavage activity of the Cas12 protein is activated when specific hybridization of the guide RNA and the target sequence occurs; iii) a third container and a nucleic acid probe detectably labeled in the third container, wherein the detectably labeled nucleic acid probe is susceptible to side-cleavage activity of the Cas12 protein; and iv) a fourth container and a buffer solution in the fourth container; wherein the target nucleic acid molecule is a target DNA.

9. Method for detecting whether a target nucleic acid molecule containing a target sequence exists in a sample, characterized in that it comprises the following steps: (a) providing the detection system for detecting a target nucleic acid molecule, as defined in claim 6, wherein the detection system further comprises a sample to be detected; and (b) detecting whether the nucleic acid probe labeled detectably in the detection system is cleaved by a Cas12 protein, wherein the cleavage is a transcleavage of a single-stranded co-stranded DNA; wherein if the nucleic acid probe labeled detectably is cleaved by the Cas12 protein, then this indicates the presence of the target nucleic acid molecule in the sample; and if the nucleic acid probe labeled detectably is not cleaved by the Cas12 protein, this indicates the absence of the target nucleic acid molecule in the sample.

10. Method according to claim 9, characterized in 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) of a target site do not have a PAM sequence, nucleic acid amplification is performed using a PAM introducer primer.

11. Method according to claim 10, characterized in that the PAM introducer primer has a structure of formula I in 5'-3': P1-P2-P3 (I) wherein, P1 is a 5' segment sequence that is located at the 5' terminus and is complementary or non-complementary to the target nucleic acid molecule sequence; P2 is a PAM sequence; and P3 is a 3' segment sequence that is located at the 3' terminus and is complementary to the target nucleic acid molecule sequence.

12. Method, according to claim 9, characterized in 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) of the target site contain the PAM sequence, then a primer containing or not containing the PAM sequence can be used and the amplified amplification product will contain the PAM sequence.

13. Use of a Cas12 protein, having collateral cleavage activity, in the preparation of a kit for detecting target nucleic acid molecules as defined in claim 8 based on collateral single-stranded DNA cleavage, characterized in that the Cas12 protein is Cas12a or a Cas12 protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a.

14. Use according to claim 13, characterized in that the Cas12 protein which has collateral single-stranded DNA cleavage activity similar to that of Cas12a is Cas12b (or C2c1). Petition 870260076986, dated 07 / 31 / 2026, p. 14 / 19

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