Application of cas protein, method for detecting target nucleic acid molecule and kit

Abstract

Provided in the present invention are an application 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 comprising: adding a guide RNA, Cas12a and a nucleic acid probe to a reaction system comprising a target nucleic acid molecule to be detected, and carrying out detection after reaction is complete.

Description

TECHNICAL FIELDThe present invention belongs to the field of biotechnology, and in particular to a method fordetecting a target nucleic acid molecule.BACKGROUNDA specific nucleic acid detection method has an important application value, such as pathogendetection, genetic disease detection, etc. In an aspect of pathogen detection, since each pathogenmicroorganism has its unique characteristic nucleic acid molecular sequence, it is possible todevelop nucleic acid molecular detection for specific species, also known as nucleic acid diagnostic(NAD), which is of great significance in the fields of food safety, environmental microorganismpollution detection, human pathogen infection, etc. Another aspect is the detection of singlenucleotide polymorphism (SNP) in human or other species. Understanding the relationshipbetween a genetic variation and a biological function at the genomic level provides a newperspective for modern molecular biology. SNP is closely related to biological functions, evolutionand diseases, so the development of SNP detection and analysis technologies is particularlyimportant.At present, many NAD methods have been established, mainly for the detection of a specificDNA molecule, and there are also some methods for RNA molecules. Generally speaking, a DNAmolecule is very stable, so a test sample can come from a series of complex biological samples;while RNA is very easy to degrade, so it needs to be handled with great care. In the 1970s, adetection method using restriction endonuclease digestion was established. Later, methods such asSouthern, Northern and dot blot hybridization were developed for specific detection of a nucleicacid molecule. In 1985, when PCR became a conventional experimental method, it led to anexponential improvement in molecular biology. Currently established specific nucleic acidmolecule detection usually needs to be performed in two steps, the first step being the amplificationof a target nucleic acid and the second step being the detection of the target nucleic acid. PCRtechnology is an amplification method that is first established and most commonly used at present.Currently, based on the PCR method, a fluorescent-labeled probe is introduced, such that theamplification situation of a target can be detected in real time, which is called Realtime PCR. TheRealtime PCR is not only a fast and highly-sensitive detection method, but also a method forquantitative analysis. In addition to the PCR amplification method, many alternative methods havebeen established, such as ligase chain reaction, branched DNA amplification, NASBA, SDA,transcription-mediated amplification, Loop-mediated isothermal amplification (LAMP), rollingcircle amplification (RCA), Recombinase Polymerase Amplification (RPA), etc. The advantage ofmany of these alternative methods is isothermality. That is to say, only one temperature is neededto complete the reaction, without the need for a thermal cycling instrument like that used in PCR.Among nucleic acid detection methods, besides the Realtime PCR that can directly completeamplification and detection, the FISH (Fluorescence in situ hybridization) technology is the mostcommonly-used detection method - a method in which a labeled molecular probe is in situhybridized with a complementary target sequence. In addition, detection methods such as nextgenerationsequencing technologies and Oxford Nanopore sequencing technologies have also beendeveloped, but these methods usually require expensive experimental equipment.The detection of SNPs also first requires amplification by a method such as PCR and the like,so as to obtain sufficient SNP site-containing region fragments for further detection. Commonlyused methods include: primer extension, hybridization, ligation, and enzymatic cleavage. Whenthe above methods are completed, a specific method needs to be utilized for detection, such asmass spectrometry detection, fluorescence detection, chemiluminescence detection, etc.Although many detection methods have been developed for nucleic acid detection as describedabove, in certain cases, how to detect more quickly, simply and less expensively is still animportant development direction, such as rapid detection of pathogenic bacteria in the field, rapiddetection of drug-sensitive SNPs, etc. In 2016, Collins et al. developed a rapid and inexpensivemethod for detecting the Zika virus based on the characteristic of CRISPR-Cas9 of specificallyrecognizing and cleaving a target sequence. In 2017, Feng Zhang et al. established a rapid nucleicacid detection method utilizing a feature of "collateral effect" of CRISPR-Cas13a. The "collateraleffect" means that Cas13a binds to a specific target RNA and then randomly cleaves other nontargetRNAs (here RNA molecules are designed as an RNA fluorescence reporting system); rapidtarget RNA detection is realize d by combining with an isothermal amplification technology RPA;and the team of Feng Zhang called this detection method as SHERLOCK (Specific HighSensitivity Enzymatic Reporter UnLOCKing). The SHERLOCK method involves the binding toan RNA template, so if DNA detection is required, the DNA needs to be firstly transcribed into anRNA template for detection; and given the instability of RNA, this method will undoubtedlyincrease the difficulty of operation.In 2015, Feng Zhang et al. discovered a new CRISPR-related endoproteinase Cas12a (formerlyknown as Cpf1), which, like a commonly used Cas9 protein, is an RNA-guided specific DNAendonuclease; however, compared with Cas9, Cas12a has its own characteristics, for example, onlya crRNA is needed to guide the specific cleavage of a double-stranded DNA and a sticky end isproduced.SUMMARYAn objective of the present invention is to provide a method for detecting a target nucleic acidmolecule.Another objective of the present invention is to provide use of a Cas protein in a method fordetecting a target nucleic acid molecule.In a first aspect of the present invention, provided is a kit including a guide RNA, a Cas protein,a nucleic acid probe, and a buffer solution.A method for detecting a target nucleic acid molecule includes adding a guide RNA, a Casprotein, a nucleic acid probe and a buffer solution into a reaction system containing a target nucleicacid molecule to be detected, and then detecting the target nucleic acid (especially by a method ofdetecting fluorescence intensity).Preferably, the Cas protein is Cas12a or Cas protein having a collateral single-stranded DNAcleavage activity similar to that of the Cas12a.Preferably, the Cas protein is Cas12a.The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a,OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a.Preferably, the Cas12a is LbCas12a.Preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind toa target DNA.In another preferred embodiment, the nucleic acid probe is a single-stranded DNA; the single10stranded DNA is preferably a fluorescently-labeled single-stranded DNA; the single-strandedDNA is preferably a fluorescent probe that is labeled with a fluorescent group HEX at a 5' terminaland labeled with a quenching group BHQ1 at a 3' terminal.In another preferred embodiment, the method for detecting the nucleic acid probe is preferablya fluorescence detection method; and the fluorescence detection method is preferably a detectionmethod using a microplate reader or a fluorescence spectrophotometer.Preferably, the target nucleic acid molecule to be detected in the reaction system of the targetnucleic acid molecule to be detected is obtained after amplification.Preferably, the detection method of the present invention can be used for detecting a pathogenicmicroorganism, gene mutation, or a specific target DNA.In another preferred embodiment, the Cas protein includes Cas12b (C2c1).In a second aspect of the present invention, provided is use of a Cas protein in a method fordetecting a target nucleic acid molecule, or in preparation of a formulation for detecting a targetnucleic acid molecule.In another preferred embodiment, when a target DNA, a guide RNA and a Cas protein form aternary complex, the complex will cleave other single-stranded DNA molecules in the system.Preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind toa target DNA.In a third aspect of the present invention, provided is a kit including a guide RNA, a Cas protein,and a nucleic acid probe.In another preferred embodiment, the kit further includes a buffer solution.In a fourth aspect of the present invention, provided is a detection system for detecting a targetnucleic acid molecule, the system including:(a) a Cas protein, which is Cas12a or a Cas protein having a collateral single-stranded DNAcleavage activity similar to that of the Cas12a;(b) a guide RNA that guides the Cas protein to specifically bind to the target nucleic acidmolecule; and(c) a nucleic acid probe which is a single-stranded DNA;wherein the target nucleic acid molecule is a target DNA.In another preferred embodiment, the detection system further includes (d) a buffer solution.In another preferred embodiment, the detection system further includes a target nucleic acidmolecule to be detected.In another preferred embodiment, the concentration of the target nucleic acid molecule to bedetected 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.In another preferred embodiment, in the detection system, the molar ratio of the nucleic acidprobe to the target nucleic acid molecule is 103: 1 to 1014: 1, and preferably 104: 1 to 107: 1.In another preferred embodiment, the detection site of the target nucleic acid molecule islocated at positions 1-12 downstream of the PAM sequence of the guide RNA.In another preferred embodiment, the length of the guide RNA is 15-30 nt, and preferably 15-18 nt.In another preferred embodiment, the target DNA includes a cDNA.In another preferred embodiment, the target DNA is selected from a group consisting of asingle-stranded DNA, a double-stranded DNA, or a combination thereof.In another preferred embodiment, the nucleic acid probe carries a fluorescent group and aquenching group.In another preferred embodiment, the fluorescent group and the quenching group are eachindependently located at the 5' terminal, 3' terminal and the middle portion of the nucleic acidprobe.In another preferred embodiment, the length of the nucleic acid probe is 3-300 nt, preferably5-100 nt, more preferably 6-50 nt, and most preferably 8-20 nt.In another preferred embodiment, the target nucleic acid molecule includes a target nucleicacid molecule derived from a group consisting of plants, animals, insects, microorganisms, viruses,or a combination thereof.In another preferred embodiment, the target DNA is an artificially-synthesized or naturallyoccurring DNA.In another preferred embodiment, the target DNA includes a wild-type or mutant DNA.In another preferred embodiment, the target DNA includes a DNA obtained by reversetranscription of RNA or amplification, such as a cDNA, etc.In another preferred embodiment, the 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, the Cas12a is LbCas12a.In another preferred embodiment, the Cas protein having a collateral single strand DNAcleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (i.e.,C2c1).In another preferred embodiment, the Cas12b protein is selected from a group consisting ofAacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus),AkCas12b (Alicyclobacillus kakegawensis), AmCas12b (Alicyclobacillus macrosporangiidus),AhCas12b (Alicyclobacillus herbarius), and AcCas12b (Alicyclobacillus contaminans).In another preferred embodiment, the nucleic acid probe comprises a single-stranded DNAcarrying a detectable label.In another preferred embodiment, the single-stranded DNA is a fluorescent-labeled and biotinlabeledsingle-stranded DNA.In another preferred embodiment, the single-stranded DNA is a fluorescent-labeled singlestrandedDNA.In another preferred embodiment, the single-stranded DNA is a fluorescent probe that is labeledwith a fluorescent group HEX at a 5' terminal and labeled with a quenching group BHQ1 at a 3'terminal.In a fifth aspect of the present invention, provided is a kit for detecting a target nucleic acidmolecule, the kit including:i) a first container and a Cas protein in the first container, the Cas protein being Cas12a or aCas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a;ii) an optional second container and a guide RNA in the second container, the guide RNAguiding the Cas protein to specifically bind to the target nucleic acid molecule;iii) a third container and a nucleic acid probe in the third container; andiv) an optional fourth container and a buffer solution located in the fourth container;wherein the target nucleic acid molecule is a target DNA.In another preferred embodiment, any two, three, or four (or all) of the first, second, third, andfourth containers may be the same or different containers.In another preferred embodiment, the nucleic acid probe carries a fluorescent group and aquenching group.In a sixth aspect of the present invention, provided is a method for detecting whether a targetnucleic acid molecule exists in a sample, including the following steps:(a) providing the detection system for detecting a target nucleic acid molecule according to thefourth aspect of the present invention, wherein the detection system further has a sample to bedetected; and(b) detecting whether the nucleic acid probe in the detection system is cleaved by a Cas protein,wherein the cleavage is a trans-cleavage of a collateral single-stranded DNA;where if the nucleic acid probe is cleaved by the Cas protein, then it indicates the presence ofthe target nucleic acid molecule in the sample; and if the nucleic acid probe is not cleaved by theCas protein, then it indicates the absence of the target nucleic acid molecule in the sample.In another preferred embodiment, the sample to be detected includes an un-amplified sampleand an amplified (or nucleic-acid-amplified) sample.In another preferred embodiment, the sample to be detected is a sample obtained byamplification.In another preferred embodiment, a method for amplifying the nucleic acid is selected from agroup consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chainreaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification,rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2.In another preferred embodiment, the PCR includes high temperature PCR, normaltemperaturePCR, and low-temperature PCR.In another preferred embodiment, the method is used for detecting whether SNP, pointmutation, deletion, and / or insertion exists in a nucleic acid at a target site.In another preferred embodiment, when the upstream and downstream (in a range of -20 nt to+20 nt, preferably in a range of -15 nt to +15 nt, and more preferably in a range of -10 nt to +10nt) of the target site lack a PAM sequence, nucleic acid amplification is carried out using a PAMintroducingprimer.In another preferred embodiment, the PAM-introducing primer has a structure of formula I in5'-3':P1-P2-P3 (I)wherein,P1 is a 5' segment sequence that is located at the 5' terminal and is complementary or non25complementary to the sequence of the target nucleic acid molecule;P2 is a PAM sequence;P3 is a 3' segment sequence that is located at the 3' terminal and is complementary to thesequence of the target nucleic acid molecule.In another preferred embodiment, the PAM primer specifically binds to upstream ordownstream of the target nucleic acid molecule.In another preferred embodiment, P1 has a length of 0-20 nt.In another preferred embodiment, P3 has a length of 5-20 nt.In another preferred embodiment, the PAM primer has a length of 18-50 nt, and preferably 20-35 nt.In another preferred embodiment, the complementation includes complete complementationand partial complementation.In another preferred embodiment, at least one primer containing the PAM sequence is used inthe nucleic acid amplification.In another preferred embodiment, when the upstream and downstream (in a range of -20 nt to+20 nt, preferably in a range of -15 nt to +15 nt, and more preferably in a range of -10 nt to +10nt) of the target site contain a PAM sequence, then a primer containing or not containing the PAMsequence can be used, and the amplified amplification product contains the PAM sequence.In another preferred embodiment, the detection in step (b) includes a fluorescence detectionmethod.In another preferred embodiment, the fluorescence detection method uses a microplate readeror a fluorescence spectrophotometer for detection.In a seventh aspect of the present invention, provided is use of a Cas protein in preparation ofa detection reagent or kit for detecting a target nucleic acid molecule based on collateral singlestrandedDNA cleavage, where the Cas protein is Cas12a or a Cas protein having a collateralsingle-stranded DNA cleavage activity similar to that of the Cas12a.In another preferred embodiment, the 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, the Cas12a is LbCas12a.In another preferred embodiment, the Cas protein having a collateral single-stranded DNAcleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (or C2c1).In another preferred embodiment, the Cas12b protein is selected from a group consisting ofAacCas12b.It should be understood that, within the scope of the present invention, the above-mentionedtechnical features of the present invention and the technical features described in detail hereafter(e.g., in examples) can be combined with each other to form a new or preferred technical solution.As limited by the length, it will not be repeated any more here.BRIEF DESCRIPTION OF DRAWINGSFIG. 1 shows a cis cleavage characteristic of Cas12a in cleaving a target single-stranded DNA.FIG. 2 shows that when cleaving a target single-stranded DNA, Cas12a does not depend on aPAM sequence required for cleaving double strands.FIG. 3 shows a trans cleavage characteristic of Cas12a in cleaving a single-stranded DNA.FIG. 4 shows test Cas12as from 10 different sources, where all of these Cas12as have cis15cleavage and trans-cleavage activities on a single-stranded DNA.FIG. 5 identifies sites possibly related to cis-cleavage and trans-cleavage activities on thesingle-stranded DNA in Cas12a through a single-site mutation experiment of the Cas12a.FIG. 6 shows the structures of Cas12a and Cas12b (i.e., C2c1) monomers and their complexeswith a guide RNA and a target DNA.FIG. 7 shows fluorescence values obtained by different Cas12as using a specific doublestrandedDNA substrate and a single-stranded DNA (HEX-N12-BHQ1) as a fluorescence detectionprobe. The negative control group is not added with the specific substrate.FIG. 8 shows a schematic flow chart of a HOLMES method for detecting a target DNA basedon target DNA amplification and the trans-cleavage activity of the Cas12a on a collateral single25stranded DNA.FIG. 9 shows a sensitivity test of a target DNA by using FnCas12a or LbCas12a directly, or incombination with the HOLMES method.FIG. 10 shows fluorescence detection values of target sequences having different single-pointmutations as detected by the HOLMES method using crRNAs of different lengths of guidesequences in combination with FnCas12a or LbCas12a.FIG. 11 tests whether a FAM-labeled single-stranded DNA probe is trans-cleaved after thetarget single-stranded DNA is added by utilizing a FAM-labeled fluorescent probe and 10 Cas12aproteins.FIG. 12 tests fluorescence values after the target single-stranded DNA is added by using HEXN12-BHQ1 as a probe and 10 Cas12a proteins.FIG. 13 (A) shows HOLMES detection values when a gyrB gene fragment is used as a targetsequence and different concentrations of pure cultured Escherichia coli MG1655 are used aspositive control templates by using a single-stranded DNA fluorescent probe labeled with HEXand BHQ1 at two ends thereof. It is shown that the fluorescence response value of Escherichia coliMG1655 decreases with the decrease of its concentration. (B) Detection values of water samplesin environments at different locations.FIG. 14 shows a schematic flow chart of a HOLMES method for detecting SNP, andfluorescence detection values of 5 SNP sites.FIG. 15 shows fluorescence detection values of key sites in a TP53 gene (a cancer-related gene)as detected by the HOLMES method.FIG. 16 shows detection values of 5 SNP sites (related to gout) as detected by the HOLMESmethod.FIG. 17 shows detection values of one SNP site (related to gout) as detected by the HOLMESmethod, where the samples are samples from 21 volunteers.FIG. 18 shows a primer design scheme of one example of the present invention, which can beused for HOLMES detection of SNP at any site.FIG. 19 uses a combination of LAMP and HOLMES to detect Escherichia coli in the system.(A) An electrophoresis map of a gyrB gene of Escherichia coli amplified by LAMP. A total of twosets of primers gyrB-1 and gyrB-2 are used for amplification. gyrB is the characteristic gene ofEscherichia coli. (B) A HOLMES detection system is used for detecting an amplification productof LAMP. Negative control: the sample is sterile water, and a gyrB-1 amplification primer is usedfor amplifying or detecting the result of the gyrB gene; gyrB-1: the sample is Escherichia coli tobe detected, and a first set of gyrB gene amplification primers are used for amplifying or detectingthe result of the gyrB gene; and gyrB-2: the sample is Escherichia coli to be detected, and a secondset of gyrB gene amplification primers are used for amplifying or detecting the result of the gyrBgene.FIG. 20 detects the genotype of a human HEK293T cell by using a combination of LAMP andHOLMES. (A) An electrophoresis map of a corresponding SNP detection template of the humanHEK293T cell as amplified by LAMP. Negative control: the sample is sterile water, and the resultis a result of amplification using an rs5082 amplification primer; rs5082: the sample is a totalgenome of the human HEK293T cell, and the result is a result of amplification using the rs5082amplification primer; and rs1467558: the sample is the total genome of the human HEK293T cell,and the result is a result of amplification using an rs1467558 amplification primer. (B) A HOLMESdetection system is used for detecting an amplification product of LAMP. The rs5082 site wasdetected using two crRNAs of crRNA-G and crRNA-T respectively (Sequence Listing 5); and thers1467558 site was detected using two crRNAs of crRNA-C and crRNA-T respectively (SequenceListing 5).FIG. 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 primersgyrB-1 and gyrB-2 are used for amplification. gyrB is a characteristic gene of Escherichia coli.(B) A HOLMES detection system is used for detecting an amplification product of RPA. Negativecontrol: the sample is sterile water, and a gyrB-1 amplification primer is used for amplifying ordetecting the result of the gyrB gene; gyrB-1: the sample is Escherichia coli to be detected, and afirst set of gyrB amplification primers are used for amplifying or detecting the result of the gyrBgene; and gyrB-2: the sample is Escherichia coli to be detected, and a second set of gyrBamplification primers are used for amplifying or detecting the result of the gyrB gene.FIG. 22 shows detection of collateral single-stranded DNA cleavage activity of Cas12b whena single-stranded DNA is used as the target DNA. After the collateral cleavage reaction iscompleted, the reactants are separated by 12% urea-denatured gel electrophoresis, and detected bya fluorescence imaging system. The numbers in brackets represent the final concentrations of thereactants in nM; the target DNA is a 66 nt long single-stranded DNA at a dosage of 50 nM; andthe single-stranded DNA probe is a single-stranded DNA carrying a FAM label at the 5' terminalat a dosage of 50 nM. As can be seen from the figure, after Cas12b, the guide RNA and the targetDNA are contained, the FAM-labeled single-stranded DNA is cut into fragments, i.e., Cas12b hasa collateral single-stranded DNA cleavage activity.FIG. 23 shows detection of collateral single-stranded DNA cleavage activity of Cas12b whena single-stranded DNA and a double-stranded DNA are used as the target DNAs. After thecollateral cleavage reaction is completed, the reactants are detected by using a fluorescencemicroplate reader. The dosages of Cas12b and the guide RNA are both 500 nM; the target DNA isa 66 nt long single-stranded DNA or double-stranded DNA at a dosage of 50 nM; and the single15stranded DNA probe is a single-stranded DNA probe (HEX-N12-BHQ1) containing a fluorescencereport group and a quenching group at a dosage of 500 nM. As can be seen from the figure,regardless of whether a single-stranded DNA template or a double-stranded DNA template is used,the collateral single-stranded DNA cleavage activity can be detected after Cas12b and the guideRNA are added.FIG. 24 shows the collateral single-stranded DNA trans-cleavage activity of Cas12b on a targetDNA at a low concentration after the combination with LAMP amplification.DETAILED DESCRIPTIONIn order to make the objectives, technical solutions and advantages of the embodiments of thepresent invention clearer, the following clearly and completely describes the technical solutions inthe embodiments of the present invention with reference to the accompanying drawings in theembodiments of the present invention. The described embodiments are a part rather than all of theembodiments of the present invention. All other embodiments obtained by a person of ordinaryskill in the art based on the embodiments of the present invention without creative efforts fallwithin the protection scope of the present invention.The inventor has developed a technical solution for target nucleic acid detection throughextensive and in-depth research and through research on the cleavage characteristics of a Casenzyme (such as Cas12a and Cas12b enzymes). The experimental results show that a nucleic acidis detected successfully and rapidly by employing the above-mentioned technical solution, forexample, for the identification of whether there is a certain concentration of microorganisms suchas Escherichia coli in water, and for the rapid identification of an SNP genotype. The presentinvention is completed on this basis.TermsThe term "guide RNA" refers to an RNA that guides a Cas protein to specifically bind to atarget DNA sequence.The term "crRNA" refers to a CRISPR RNA, which is a short RNA that guides Cas12a to bindto a target DNA sequence.The term "CRISPR" refers to a clustered regularly interspaced short palindromic repeat, whichis the immune system of many prokaryotes.The term "Cas protein" refers to a CRISPR-associated protein, which is a related protein in aCRISPR system.The term "Cas12a" (formerly referred to as "Cpf1") refers to a crRNA-dependent endonuclease,which is a type V-A enzyme in CRISPR system classification.The terms "Cas12b" and "C2c1" are used interchangeably, and refer to a crRNA-dependentendonuclease, which is a type V-B enzyme in CRISPR system classification.The term "LAMP" is a Loop-mediated isothermal amplification technology, which is anisothermal nucleic acid amplification technology suitable for gene diagnosis.The term "PAM" refers to a protospacer-adjacent motif, which is necessary for Cas12acleavage. The PAM of FnCas12a is a TTN sequence, the PAM of LbCas12a is a TTTN sequence,and the PAM of AacCas12b is TTN.The present invention discloses a method for detecting a target nucleic acid molecule, whichincludes: adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution into areaction system containing a target nucleic acid molecule to be detected, and then performingfluorescence detection of the target nucleic acid molecule.The Cas protein is Cas12a or Cas12b.The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a,OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; and the Cas12a is preferablyLbCas12a.The Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b orAcCas12b.A guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.The target nucleic acid molecule to be detected in the reaction system of the target nucleic acidmolecule to be detected is obtained by amplification.The detection method can detect a pathogenic microorganism, gene mutation, or a specifictarget DNA.Use of a Cas protein in a method for detecting a target nucleic acid molecule.When a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex willcleave other single-stranded DNA molecules in the system.A guide RNA refers to an RNA that guides a Cas protein to specifically target a DNA sequence.The present invention also provides a kit including a guide RNA, a Cas protein, and a nucleicacid probe. Furthermore, the kit of the present invention can also include a buffer solution.The present invention provides a detection method for rapidly detecting a target nucleic acidmolecule with high specificity. Once the (single-stranded or double-stranded) target DNA, thecrRNA and the Cas12a protein form a ternary complex, the complex will cleave other singlestrandedDNA molecules in the system. Through designing, the crRNA targets the target DNA (asegment of DNA sequence to be detected); the crRNA and the Cas12a protein are added into thedetection system; when the target DNA is present, the Cas12a forms a ternary complex with thecrRNA and the target DNA, and meanwhile, the complex exerts its collateral cleavage activity andcleaves a single-stranded DNA labeled with a fluorescent signal (two ends of the single-strandedDNA are respectively connected with a luminescent group and a quenching group, and theluminescent group can emit light after being cleaved), thereby emitting fluorescence. Therefore,whether the system to be detected contains the target DNA molecule can be known by fluorescencedetection. By using the method of the present invention, whether a specific DNA sequence iscontained in a sample can be quickly detected. The sensitivity of the detection method can begreatly improved by combining with a PCR technology. The nucleic acid probe in the presentinvention is preferably a fluorescent probe.HOLMES condition testing:The present invention provides use of Cas12 enzymes such as Cas12a and Cas12b in nucleicacid detection. The following description will take Cas12a as an example.Selection of Cas12a: according to research, Cas12a has a trans-cleavage activity, that is, oncethe target DNA, the crRNA and the Cas12a protein form a ternary complex, other single-strandedDNAs (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 fluorescentprobe, which consists of a random sequence of 12 nt, and is labeled with a fluorescent group HEXat the 5' terminal and a quenching group BHQ1 (HEX-N12-BHQ1) at the 3' terminal. When thetarget DNA fragment is contained in the system, a ternary complex of the target DNA, the crRNAand the Cas12a protein will be formed. At this time, the probe will be cleaved, and meanwhile, theHEX fluorescent group will emit fluorescence (with an excitation light at 535 nM and an emissionlight at 556 nM) as detected by a fluorescence detector. Next, 10 different Cas12as are tested, andthe target sequence is a double-stranded DNA as shown in FIG. 7. It can be seen that the complexcomposed of the target double-stranded DNA and each Cas12a protein can realize the transcleavage activity.HOLMES response sensitivity: then, the response sensitivities of FnCas12a and LbCas12ato the target DNA are tested, that is to say, the lowest concentration of the target DNA at whichthe response can occur is investigated. As shown in FIG. 9, when the test target is directly added,they can respond to the target DNA with a concentration above 0.1 nM, and the response isremarkable when the concentration is above 1 nM. If a PCR technology (the HOLMES method) iscombined, as shown in FIG. 8, i.e., amplification of the fragment of interest through PCR followedby a Cas12a cleavage reaction, the response sensitivity can be as low as 10 aM, as shown in FIG.9.SNP Test: next, whether the HOLMES method can detect an SNP genotype is tested. T1 istaken as a target sequence, PAM at this site is mutated or positions 1-18 of the target sequence arerespectively subjected to single-point mutation, and the detection differences between a nonmutatedsequence and a mutated sequence by crRNAs of different lengths are compared.As shown in FIG. 10, when the complementary sequence of the target is a crRNA of 24 nt(crRNA-24nt), the single-point mutation at the positions 8-18 is not much different from the wildtype, while the fluorescence value decreases obviously after the PAM mutation and the mutationof positions 1-7. When the crRNA is truncated and the length of a paired target sequence is 18 nt,the fluorescence value of the mutation positions of 8-16 nt is obviously decreased compared withthat of a target sequence having a length of 24 nt; when the length of the crRNA continues to beshortened to 16 nt or 17 nt, the fluorescence value of the mutated target sequence decreases moresignificantly; and when it is further shortened to 15 nt, the fluorescence value of the mutated targetsequence is weaker as compared with that of this target sequence, but the fluorescence intensity ofthe mutated target sequence may still be higher as compared with those of other target sequences,and thus can be used for detection. Taken together, the crRNAs of 15 nt, 16 nt and 17 nt are themost suitable for SNP detection.In the present invention, the Cas12a cleaves the single-stranded DNA in a PAM-sequenceindependent programmed cleaving manner, which is called cis cleavage; but once the ternarycomplex Cas12a / crRNA / target DNA is formed, it will show a trans-cleavage activity, i.e.,exhibiting an activity of cleaving any non-target single-stranded DNA in the system.Based on the characteristics of Cas12a, a method for specific nucleic acid molecule detectionis developed, which is called HOLMES (one HOur Low-cost Multipurpose Efficient Simpleassay). Just like the name of the technology, it is characterized by a fast speed (1 hour), a low price,multiple channels, high efficiency and a simple test method. The method can be used in the fieldsof rapid pathogen detection, SNP detection and the like.Nucleic acid detection based on the collateral cleavage activityThe present invention also provides a nucleic acid detection method based on the collateralcleavage activity of the Cas12 enzyme (including Cas12a or Cas12b).Preferably, the detection of the present invention can be performed for SNP, and in particularPCR amplification is performed first followed by detection.Referring to FIG. 18, a primer design scheme is given.Case 1. When there is a PAM site near the SNP site, and a crRNA synthesized based on a guidesequence designed according to the PAM site can be used for HOLMES detection. When theHOLMES method is used for detection, it shows a relatively low background signal; and for thesame guide sequence, signal differences between different SNP templates are quite large.Case 2. When there is no PAM site or no suitable PAM site near the SNP site, a PAM site canbe introduced according to the above experimental scheme.A typical step includes designing a primer near the SNP site, and carrying a PAM site on theprimer, where the sequence located at the 3' terminal of the PAM site should be paired with thetemplate DNA. There is no special requirement on the primer at the other terminal, as long as theprimer can be paired with template DNA and can be used for PCR amplification. As shown in FIG.18, PAM sites can be successfully introduced after PCR amplification.Referring to FIG. 10, in the present invention, when designing introduction of the PAM site,the SNP site is usually located at the first 16 bases of the 5' terminal of the crRNA guide sequence,preferably at positions 1-14, more preferably at positions 1-12, still more preferably at positions 1-11 or 1-10, and most preferably at positions 1-8 or 1-7.The present invention has the following main advantages:(1) Fast speed: when the test conditions are ready, it only takes about 1 hour from the time ofobtaining a sample to the time of obtaining test results.(2) Low cost: there are no special materials or enzymes in the experiment, and it involves asmall quantity of materials and reagents, and thus it can be used for trace analysis.(3) High efficiency: the present invention has extremely high sensitivity and can detect DNAat a concentration of 10 aM.(4) Multiple uses: it can detect different nucleic acid samples, including DNA samples andRNA samples.(5) Simplicity: there are no special or complicated steps, and if a kit is prepared and a programis set, only simple operations such as adding a sample are required.The present invention will be described in detail below in connection with specific examples.It should be understood that the following examples are only intended to illustrate the presentinvention, rather than limiting the scope of the present invention. The experimental methods in thefollowing examples which are not specified with specific conditions are generally carried outaccording to conventional conditions, such as those described in Sambrook et al., MolecularCloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or thoserecommended by manufacturers. Unless otherwise stated, percentages and parts are weightpercentages and parts by weight.Unless otherwise stated specifically, the experimental materials involved in the presentinvention can be obtained from commercial channels.Materials1. The RNase inhibitor is purchased from TaKaRa, and the high-fidelity DNA polymeraseKOD FX is purchased from ToYoBo; the primers (oligonucleotides) are synthesized by SangonBiotech (Shanghai) Co., Ltd.; the T7 RNA polymerase is purchased from Thermo; the RNApurification and concentration kit (RNA Clean&ConcentratorTM_5) is purchased from ZymoResearch; the Wizard SV Gel and PCR Clean-Up System is purchased from Promega; and themedia (e.g., Tryptone, Yeast Extract, etc.) are all purchased from OXOID.2. Medium formula: a liquid LB (1% Tryptone, 0.5% Yeast extract, 1% NaCl), and only 2%agar needs to be added into the liquid LB when a solid LB is prepared.Example 1 Cas12a protein detection capable of detecting a single-stranded DNA target(the probe being labeled with FAM)A single-stranded DNA (target-T1-R) was selected as a target sequence to test the responsevalues of its detection by different Cas12a proteins.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-T1-24-R as shown in Table 5. Specifically, the pairedoligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of50 μL, and then subjected to an annealing procedure: initially denaturing at 95°C for 5 minutes,and then cooling from 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA wassynthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnightat 37°C (for about 16 h). Then RNA was purified using the RNA purification and concentrationkit, quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted to a concentration of 10μM, and stored in a refrigerator at -80°C.2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), a target single-stranded DNA (target-T1-R) (0.01 μM), a nucleic acidprobe (N25-5' FAM) (0.01 μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of an RNaseinhibitor. The blank control reaction was a reaction in which all other components were addedexcept the single-stranded DNA target sequence. Reaction was conducted at 37°C for 15 min, andthen terminated at 98°C for 2 min.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 FIG. 11,different Cas12as have different detection effects on the target. For example, for HkCas12a, etc.,cleavage of the probe was caused even when no target single-stranded DNA was added. LbCas12aand the like are better candidates of Cas12a proteins because the cleavage of the probe occurredonly when the target single-stranded DNA was added.Example 2 Cas12a protein detection capable of detecting a single-stranded DNA target(the probe being labelled with two labels of HEX and BHQ1)A single-stranded DNA (target-T1-R) was selected as a target sequence to test the responsevalues of its detection by different Cas12a proteins.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide(4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and thensubjected to an annealing procedure: initially denaturing at 95°C for 5 minutes, and then coolingfrom 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesizedusing a T7 high-throughput transcription kit, and the reaction was carried out overnight at 37°C(for about 16 h). Then RNA was purified using the RNA purification and concentration kit,quantified with NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigeratorat -80°C.2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), a target single-stranded DNA (target-T1-R) (0.01 μM), a fluorescenceprobe (HEX-N12-BHQ1, i.e., a 12 nt single-stranded DNA that is labeled with HEX at the 5'terminal and with the BHQ1 at the 3' terminal) (0.5 μM), a buffer solution of NEB buffer 3.1, and0.5 μL of an RNase inhibitor. The control reaction was a reaction in which all other componentswere added except the single-stranded DNA target sequence. Reaction was conducted at 37°C for15 min, and then terminated at 98°C for 2 min.3. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 12, different Cas12as have different detection effects on thetarget. For example, for HkCas12a, etc., cleavage of the probe was caused even when no targetsingle-stranded DNA was added. FnCas12a and the like are better candidates of Cas12a proteinsbecause the cleavage of the probe occurred only when the target single-stranded DNA was added.Example 3 Cas12a protein detection capable of detecting a double-stranded DNA targetA double-strand DNA (target-T1) was selected as a target sequence to test the response valuesof its detection by different Cas12a proteins.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide(4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and thensubjected to an annealing procedure: initially denaturing at 95°C for 5 minutes, and then coolingfrom 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesizedusing a T7 high-throughput transcription kit, and the reaction was carried out overnight at 37°C(for about 16 h). Then RNA was purified using the RNA purification and concentration kit,quantified with NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigeratorat -80°C.2. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), a target double-stranded DNA (target-T1, obtained by annealingprimers target-T1-F to target-T1-R) (0.01 μM), a fluorescence probe (HEX-N12-BHQ1) (0.5 μM),a buffer solution of NEB buffer 3.1, and 0.5 μL of an RNase inhibitor. Reaction was conducted at37°C for 15 min, and then terminated at 98°C for 2 min.3. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 7, different Cas12as have different detection effects on thetarget. LbCas12a and the like are better candidates of Cas12a proteins because the cleavage of theprobe occurred only when the target double-stranded DNA was added.Example 4 Testing of different concentrations of the target with FnCas12a and LbCas12atarget-T1 was selected as the target DNA, and then diluted to different concentrations at agradient to test the response sensitivity of FnCas12a and LbCas12a thereto. In order to increase thesensitivity, a PCR amplification step was added.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide(4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and thensubjected to an annealing procedure: initially denaturing at 95°C for 5 minutes, and then coolingfrom 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesizedusing a T7 high-throughput transcription kit, and the reaction was carried out overnight at 37°C(for about 16 h). Then RNA was purified using the RNA purification and concentration kit,quantified with NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigeratorat -80°C.2. PCR amplification (optional): a plasmid containing the target target-T1 (pUC18-T1) wasused as the template, diluted at a gradient, and then used for the PCR reaction. The total volume ofeach reaction system was 20 μL, 0.25 μM of M13F-47 and M13R-48 each were used as primers(Table 4), and a high fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCRreaction procedure was 95°C for 2 min, and then starting 35 cycles of 98°C for 10 s, 60°C for 15s, and 68°C for 10 s. After completion of the PCR, the PCR amplification product was directlyused for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, FnCas12a or LbCas12a (0.25 μM), 1 μL of a PCR product (or target DNAs that are directlydiluted into different concentrations), a fluorescent probe (HEX-N12-BHQ1) (0.5 μM), a buffersolution of NEB buffer 3.1, and 0.5 μL of a RNase inhibitor. Reaction was conducted at 37°C for15 min, and then terminated at 98°C for 2 min.4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 9, when the test target was directly added, all the target DNAswith a concentration above 0.1 nM could respond, and the response was remarkable when theconcentration was above 1 nM. If a PCR technology was combined, i.e., amplification of thefragment of interest through PCR followed by a Cas12a cleavage reaction, the response sensitivitycould be as low as 10 aM.Example 5 Testing of single-point mutant target with FnCas12a and LbCas12atarget-T1 was selected as the target, and it was subjected to single-point mutation in a PAMregion and positions 1-18 respectively, so as to test response values of several crRNAs of differentlengths to the wild type and the same after single-point mutation.1. Preparation of crRNA: first, a transcription template was prepared by respectively annealingT7-crRNA-F to 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 a1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and then subjected to an annealingprocedure: initially denaturing at 95°C for 5 minutes, and then cooling from 95°C to 20°C at a rateof 1°C per minute using a thermal cycler. crRNA was synthesized using a T7 high-throughputtranscription kit, and the reaction was carried out overnight at 37°C (for about 16 h). Then RNAwas purified using the RNA purification and concentration kit, quantified with NanoDrop 2000C,diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.2. PCR amplification: a plasmid containing the target target-T1 (pUC18-T1) was used as thetemplate. The total volume of each reaction system was 20 μL, 0.25 μM of the primer M13R-48and respective mutation primers for Target-T1-F each were used (Table 4), and a high fidelityenzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was 95°Cfor 2min, and then starting 35 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 10 s. Aftercompletion of the PCR, the product was directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, FnCas12a or LbCas12a (0.25 μM), 1 μL of a PCR product, a fluorescent probe (HEX-N12-BHQ1) (0.5 μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of a RNase inhibitor. Reactionwas conducted at 37°C for 15 min, and then terminated at 98°C for 2 min.4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 10, when the complementary sequence of the target was acrRNA of 24 nt (crRNA-24nt), the single-point mutations at the positions 8-18 were not muchdifferent from the wild type, while the fluorescence value decreased obviously after the PAMmutation and the point mutation of positions 1-7. When the crRNA was truncated and the lengthof a paired target sequence was 18 nt, the fluorescence value of the mutation positions of 8-16 ntwas obviously decreased compared with that of 24 nt; when the length was 16 nt or 17 nt, thedecrease of the fluorescence value of the mutated target sequence is more obvious; and when thelength was 15 nt, the fluorescence values of both the target sequence and the mutated targetsequence were very weak, but the fluorescence intensity of the mutated target sequence might stillbe higher as compared with those of other target sequences, and thus could be used for detection.Taken together, the crRNAs of 15 nt, 16 nt and 17 nt are the most suitable for SNP detection.Example 6 Testing of Escherichia coli and the like microorganisms in environmentalwaterThe gyrB gene of Escherichia coli was selected as the detection target to indirectly test theconcentrations of Escherichia coli and the like microorganisms in water. Taking Escherichia coliMG1655 as a positive control, the content of microorganisms in water (such as sewage and tapwater) in the environment was determined.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the pairedoligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of50 μL, and then subjected to an annealing procedure: initially denaturing at 95°C for 5 minutes,and then cooling from 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA wassynthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnightat 37°C (for about 16 h). Then RNA was purified using the RNA purification and concentrationkit, quantified with NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in arefrigerator at -80°C.2. PCR amplification: when the positive control sample Escherichia coliMG1655 was cultureduntil the OD600 was about 0.5, it was diluted at a 10-fold gradient respectively, and then used asthe template, and the sample was ambient water (including tap water and muddy water in theenvironment). The total volume of each reaction system was 20 μL, 0.25 μM of primers gyrB-Fand gyrB-R each were used (Table 4), and a high fidelity enzyme KOD FX (ToYoBo) was usedfor the PCR reaction. The PCR reaction procedure was 95°C for 2 min, and then starting 35 cyclesof 98°C for 10 s, 60°C for 15 s, and 68°C for 10 s. After completion of the PCR, the PCR productwas directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0. 5 μM) purified instep 1, LbCas12a (0.25 μM), 1 μL of a PCR product, a fluorescence probe (HEX-N12-BHQ1) (0.5μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of an RNase inhibitor. Reaction wasconducted at 37°C for 15 min, and then terminated at 98°C for 2 min .4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 13, the fluorescence response value of Escherichia coliMG1655 decreases with the concentration decrease. Among them, microorganisms were detectedmore obviously in samples 2, 4, 5 and 6.Example 7 Testing of Human SNPThe SNP test selected 5 sites of human SNP, namely rs5082, rs1467558, rs2952768,rs4363657, and rs601338, to test the feasibility of the HOLMES method.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) wasannealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and then subjected toan annealing procedure: initially denaturing at 95°C for 5 minutes, and then cooling from 95°C to20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesized using a T7 highthroughputtranscription kit, and the reaction was carried out overnight at 37°C (for about 16 h).RNA was purified using the RNA Clean &Concentrator-5 (Zymo Research), quantified withNanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of primerseach were used (table 4), 1 ng of a human genome (HEK293T) or directly-scraped oral epithelialmucosa was used as the template, and a high fidelity enzyme KOD FX (ToYoBo) was used for thePCR reaction. The PCR reaction procedure was 95°C for 2 min, and then starting 35 cycles of98°C for 10 s, 60°C for 15 s, and 68°C for 10 s. After completion of the PCR, the product wasdirectly used for a Cas12a reaction. (Primers 1-rs5082-F-T, 2-rs1467558-F-T, and 3-rs2952768-R-C were directly introduced to the corresponding mutation products of the SNP)3. Cas12a reaction: a 20 μL reaction system was added with corresponding crRNA (1 μM),LbCas12a (0.5 μM), 1 μL of a PCR product, and a fluorescent probe (HEX-N12-BHQ1) (0.5 μM).Reaction was conducted at 37°C for 15 min, and then terminated at 98°C for 2 min .4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 14, only when the crRNA corresponded to the correspondingtarget sequence, there would be a higher fluorescence response value, and if there was one pointmutation, its response value would be greatly reduced. The genotype of the corresponding SNPcould be determined by the fluorescence value, and these results were confirmed by sequencingresults.Example 8 Testing of a cancer-related geneA TP53 gene was selected as the test gene. TP53 gene has a nonsense mutation in a human T24cell, which leads to inactivation of the gene. A cell with normal gene at this site (HEK293T), anindividual gene and a mutant cell T24 were tested respectively.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNA20F to synthesized oligonucleotides T7-crRNA-34-TP53-T24-C-16nt and T7-crRNA-34-TP53-T24-G-16nt (Table 5). Specifically, the paired oligonucleotide (4 μM) was annealed in a 1X PCR buffer(Transgen Biotech) at a total volume of 50 μL, and then subjected to an annealing procedure:initially denaturing at 95°C for 5 minutes, and then cooling from 95°C to 20°C at a rate of 1°C perminute using a thermal cycler. crRNA was synthesized using a T7 high-throughput transcriptionkit, and the reaction was carried out overnight at 37°C (for about 16 h). RNA was purified usingthe RNA Clean &Concentrator-5 (Zymo Research), quantified with NanoDrop 2000C, dilutedto a concentration of 10 μM, and stored in a refrigerator at -80°C.2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of primers34-TP53-T24-F and 34-TP53-T24-R each were used (Table 4), 1 ng of a human genome(HEK293T, T24) or directly-scraped oral epithelial mucosa was used as the template, and a highfidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedurewas 95°C for 2 min, and then starting 35 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 10 s.After completion of the PCR, the product was directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with corresponding crRNA (1 μM),LbCas12a (0.5 μM), 1 μL of a PCR product, and a fluorescent probe (HEX-N12-BHQ1) (0.5 μM).Reaction was conducted at 37°C for 15 min, and then terminated at 98°C for 2 min .4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nM and an emissionlight at 556 nM). As shown in FIG. 15, when the TP53 gene that was normal at this site was thetemplate, the detected value of crRNA-C was significantly higher than that of crRNA-G, while thecrRNA-G of the mutant cell T24 was significantly increased.Example 9 Testing of Human SNP (Gout-Related Genes)The SNP test selected 5 sites of human SNP that were related to a gout risk, namely rs1014290,rs6449213, rs737267, rs1260326, and rs642803, to test the HOLMES method.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) wasannealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and then subjected toan annealing procedure: initially denaturing at 95°C for 5 minutes, and then cooling from 95°C to20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesized using a T7 highthroughputtranscription kit, and the reaction was carried out overnight at 37°C (for about 16 h).RNA was purified using the RNA Clean &ConcentratorTM-5 (Zymo Research), quantified withNanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.2. PCR amplification: the total volume of the reaction system was 20 μL, 0.25 μM of primerseach were used (Table 4), 1 ng of a human genome (HEK293T) or directly-scraped oral epithelialmucosa was used as the template, and a high fidelity enzyme KOD FX (ToYoBo) was used for thePCR reaction. The PCR reaction procedure was 95°C for 2 min, and then starting 35 cycles of98°C for 10 s, 60°C for 15 s, and 68°C for 10 s. After completion of the PCR, the product wasdirectly used for a Cas12a reaction. (Primers 1-rs5082-F-T, 2-rs1467558-F-T, and 3-rs2952768-R-C were directly introduced to the corresponding mutation products of the SNP)3. Cas12a reaction: a 20 μL reaction system was added with the corresponding crRNA (1 μM),LbCas12a (0.5 μM), 1 μL of a PCR product, and a fluorescence probe (HEX-N12-BHQ1) (0.5μM). Reaction was conducted at 37°C for 15 min, and then terminated at 98°C for 2 min .4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). As shown in FIG. 16, only when the crRNA corresponded to the correspondingtarget sequence, there would be a higher fluorescence response value, and if there was one pointmutation, its response value would be greatly reduced. The genotype of the corresponding SNPcould be determined by the fluorescence value, and these results were confirmed by sequencingresults.Example 10 SNP Testing of clinical samples of volunteers (a gout related gene) by a kitA pre-mixed solution was added into a 96-well plate to prepare a kit, and then the kit was addedwith genomic DNAs of 21 volunteers to test the rs1014290 site, which was related to a gout risk.1. Preparation of a kit: first, a transcription template was prepared by annealing T7-crRNA-Fto a synthesized oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 μM) wasannealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 50 μL, and then subjected toan annealing procedure: initially denaturing at 95°C for 5 minutes, and then cooling from 95°C to20°C at a rate of 1°C per minute using a thermal cycler. crRNA was synthesized using a T7 highthroughputtranscription kit, and the reaction was carried out overnight at 37°C (for about 16 h).RNA was purified using the RNA Clean &ConcentratorTM-5 (Zymo Research), quantified withNanoDrop 2000C, and diluted to a concentration of 10 μM.2. Pre-mixing in a 96-well plate for PCR: a 19 μL system was added with reagents required fora PCR reaction, the primers being 41-rs1014290-F and 41-rs1014290-R.3. Pre-mixing in a 96-well plate for fluorescence detection: the 19 μL system was added witha crRNA (1 μM), LbCas12a (0.5 μm) and a fluorescence probe (HEX-N12-BHQ1) (0.5 μM) andwas added in the 96-well plate.4. PCR amplification: the pre-mixed 96-well plate for PCR was added with the genomic DNAsof the volunteers, and then subjected to the PCR reaction. The PCR reaction procedure was 95°Cfor 2min, and then starting 35 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 10 s.5. Cas12a reaction: 1 μL of a PCR reaction solution was taken and added into the pre-mixed96-well plate for fluorescence detection, reacted at 37°C for 15 min, and then the reaction wasterminated at 98°C for 2 min.6. Fluorescence detection: it was detected by a microplate reader (with an excitation light at535 nm and an emission light at 556 nm). As shown in FIG. 17, because a population with agenotype A:A had a higher risk of gout, people other than volunteers Nos. 5, 7 and 9 are of thegenotype A:G or G:G, so more attention should be paid to the risk of gout.Example 11 Detection of Escherichia coli and the like microorganisms in environmentalwater by LAMP combined with a Cas proteinThe gyrB gene of Escherichia coli was selected as the detection target to indirectly test whetherEscherichia coli or the like microorganisms exist in water.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNA20F to a synthesized oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the pairedoligonucleotide (4 μM) was annealed in a 1 X Taq DNA polymerase reaction buffer (TransgenBiotech) at a total volume of 50 μL, and then subjected to an annealing procedure: initiallydenaturing at 95°C for 5 minutes, and then cooling from 95°C to 20°C at a rate of 1°C per minuteusing a thermal cycler. crRNA was synthesized using a T7 high-throughput transcription kit, andthe reaction was carried out overnight at 37°C (for about 16 h). Then RNA was purified using theRNA purification and concentration kit, quantified with NanoDrop 2000C, finally diluted to aconcentration of 10 μM, and stored in a refrigerator at -80°C for later use.2. LAMP amplification: sterile water and a contaminated liquid containing Escherichia coliwere used as a negative control and a sample to be detected respectively. The total volume of eachreaction system was 25 μL, primers of 1.6 μM of LAMP-FIP and LAMP-BIP each, 0.2 μM ofLAMP-F3 and LAMP-B3 each, 0.4 μM of LAMP-LoopF and LAMP-LoopB each were used, andthe kit used for the LAMP reaction was a WarmStart LAMP Kit (NEB). The LAMP reactionprocedure was 65°C for 30 min. After the LAMP was completed, annealing was conducted at 80°Cfor 10 min, and then the product was directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), 1 μL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1)(0.5 μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of a RNase inhibitor. Reaction wasconducted at 37°C for 15 min.4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). The results were as shown in FIG. 19.Example 12 Detecting SNP by using LAMP amplification combined with a Cas protein1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-crRNA-rs5082-T / T7-crRNA-rs5082-G / T7-crRNArs1467558-T / T7-crRNA-rs14 67558-C (Table 5). Specifically, the paired oligonucleotide (4 μM)was annealed in a 1 X Taq DNA polymerase reaction buffer (Transgen Biotech) at a total volumeof 50 μL, and then subjected to an annealing procedure: initially denaturing at 95°C for 5 minutes,and then cooling from 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA wassynthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnightat 37°C (for about 16 h). Then RNA was purified using the RNA purification and concentrationkit, quantified with NanoDrop 2000C, finally diluted to a concentration of 10 μM, and stored in arefrigerator at -80°C for later use.2. LAMP amplification: a human genome HEK293T was taken as a sample. The total volumeof each reaction system was 25 μL, primers of 1.6 μM of LAMP-FIP and LAMP-BIP each, 0.2 μMof LAMP-F3 and LAMP-B3 each, 0.4 μM of LAMP-LoopF and LAMP-LoopB each were used,and a WarmStart LAMP Kit (NEB) was used for a LAMP reaction. The LAMP reactionprocedure was 65°C for 30 min. After the LAMP was completed, annealing was conducted at 80°Cfor 10 min, and then the product was directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), 1 μL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1)(0.5 μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of a RNase inhibitor. Reaction wasconducted at 37°C for 15 min.4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). The results were as shown in FIG. 20.Example 13 Detection of Escherichia coli and the like microorganisms in environmentalwater by RPA amplification combined with a Cas proteinThe gyrB gene of Escherichia coli was selected as the detection target to indirectly test whetherEscherichia coli and the like microorganisms exist in water.1. Preparation of crRNA: first, a transcription template was prepared by annealing T7-crRNAFto a synthesized oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the pairedoligonucleotide (4 μM) was annealed in a 1X PCR buffer (Transgen Biotech) at a total volume of50 μL, and then subjected to an annealing procedure: initially denaturing at 95°C for 5 minutes,and then cooling from 95°C to 20°C at a rate of 1°C per minute using a thermal cycler. crRNA wassynthesized using a T7 high-throughput transcription kit, and the reaction was carried out overnightat 37°C (for about 16 h). Then RNA was purified using the RNA purification and concentrationkit, quantified with NanoDrop2000C, finally diluted to a concentration of 10 μM, and stored in arefrigerator at -80°C for later use.2. RPA amplification: sterile water and a contaminated liquid containing Escherichia coli wereused as a negative control and a sample to be detected respectively. The total volume of eachreaction system was 25 μL, 0.5 μM of primers RPA-gyrB-F (or RPA-gyrB-F2) and RPA-gyrB-R2each were used, and a TwistAmp Basic kit (TwistDX) was used for the RPA reaction. The RPAreaction procedure was 37°C for 30 min. After the RPA was completed, annealing was conductedat 80°C for 10 min, and then the product was directly used for a Cas12a reaction.3. Cas12a reaction: a 20 μL reaction system was added with the crRNA (0.5 μM) purified instep 1, a Cas12a (0.25 μM), 1 μL of a RPA product, a fluorescence probe (HEX-N12-BHQ1) (0.5μM), a buffer solution of NEB buffer 3.1, and 0.5 μL of a RNase inhibitor. Reaction was conductedat 37°C for 15 min.4. Fluorescence detection: 20 μL of the inactivated reaction solution was added into a 96-wellplate, and then detected by a microplate reader (with an excitation light at 535 nm and an emissionlight at 556 nm). The results were as shown in FIG. 21.Example 14: Cas12b having a collateral cleavage activity1. Preparation of a guide RNA (sgRNA)Firstly, a plasmid pUC18-guide RNA-T1 was constructed by using pUC18 as the skeleton ofthe plasmid. In the plasmid, a T7 promoter and a template DNA sequence for transcription of theguide RNA were inserted on the pUC18 (Note: the guide RNA transcribed from this templatetargeted a sequence called T1 in this study). The method was firstly carrying out a round of PCRby using the pUC18 plasmid as a template and PUC18-1-F and pUC18-1-R as primers; linkingPCR products with a T4 DNA Ligase, transforming the product into DH10b, and sequencing toobtain the correct clone, which was called pUC18-guide RNA-T1-pre. Then, a second round ofPCR was carried out by using the pUC18-guide RNA-T1-pre as a template and pUC18-2-F andpUC18-2-R as primers, linking and transforming the PCR products in the same way, to finallyobtain the plasmid pUC18-guide RNA-T1 which was correct as sequenced.Next, by using the plasmid pUC18-guide RNA-T1 as a template, a guide RNA was synthesizedusing a T7 high-throughput transcription kit (Thermo), and the reaction was carried out overnightat 37°C (for 12-16 h).Finally, the transcription system was added with a DNase I (2 μL of the DNase I was addedper 50 μL of the transcription system), subjected to a water bath at 37°C for 30 min to eliminate aplasmid DNA, and the RNA was purified using the RNA purification and concentration kit,quantified with the NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in arefrigerator at -80°C for later use.2. Preparation of a Target DNA(1) If the target DNA was a single strand, a 66 bp long oligonucleotide was directly synthesizedas the target DNA (target-T1-R), which contained the 20 bp target sequence (T1) recognized bythe guide RNA.(2) If the target DNA was double-stranded, two 66 bp long complementary oligonucleotides(target-T1-F; target-T1-R) were directly synthesized, which contained the 20 bp target sequence(T1) identified by the guide RNA. The two oligonucleotides were annealed to obtain a short targetDNA. Specifically, the paired oligonucleotide (1 μM) was annealed in a 1X PCR buffer (TransgenBiotech) at a total volume of 20 μL, and then subjected to an annealing procedure: initiallydenaturing at 95°C for 5 minutes, and then cooling from 95°C to 20°C at a rate of 1°C per minuteusing a thermal cycler.3. Cas12b reaction(1) annealing of the guide RNA: the guide RNA was diluted to an appropriate concentration(10 μM), and annealed in the PCR instrument. The annealing procedure: denaturing at 75°C for 5min, and then cooling from 75°C to 20°C at a decreasing rate of 1°C per minute.(2) incubation of the guide RNA with C2c1: the annealed guide RNA was mixed with C2c1 atan equal molar concentration, and allowed to stand at 30°C for 20-30 min.(3) Cas12b reaction: a 20 μL reaction system was added with a mixture of the guide RNA andC2c1 incubated in step (2) (the final concentrations of them were both 250 μM or 500 μM), a targetDNA (with a final concentration of 50 nM), a FAM-labeled oligonucleotide (target-DNMT1-3-RFAM-5') or a fluorescence quenching probe (HEX-N12-BHQ1 with the final concentration of 500nM), 2 μL of a 10 X NEB Buffer 3.1, and 0.5 μL of an RNase inhibitor (40 U / μL). After mixinghomogeneously, the reaction was conducted at 48°C for 30 min. After that, it was inactivated byheating at 98°C for 5 min in a PCR instrument.4. Detection of trans-cleavage activity of Cas12b by urea-denatured gel electrophoresis: 20 μLof the inactivated reaction solution was separated by a urea-denatured gel electrophoresis method,and then imaged by a fluorescence imaging system ImageQuant LAS 4000 mini (GE Healthcare).The results were as shown in FIG. 22.5. Detection of trans-cleavage activity of Cas12b by a fluorescence microplate reader method:20 μL of the inactivated reaction solution was added into a 96-well plate, and detected by amicroplate reader (with an excitation light at 535 nm and an emission light at 556 nm). The resultswere as shown in FIG. 23.Example 15: Sensitivity testing of a Cas12b reaction (trans-cleavage)By detecting the excited fluorescence intensity of the fluorescence probe (HEX-N12-BHQ1),the target DNA concentration required for Cas12b to exert the trans-cleavage activity, i.e., thesensitivity of a Cas12b trans-cleavage reaction, was determined.1. Preparation of a guide RNAFirstly, by using the pUC18-guide RNA-T1 as a template and guide RNA-DNMT1-3-F andguide RNA-DNMT1-3-R as primers, 20 bases of the guide RNA that target the target DNA of T1were replaced by a guide RNA targeting DNMT1-3 through PCR, so as to obtain another plasmidpUC18-guide RNA-DNMT1-3.Then, by using the plasmid pUC18-guide RNA-DNMT1-3 as a template, a guide RNA wassynthesized using a T7 high-throughput transcription kit (Thermo), and the reaction was carriedout overnight at 37°C (for 12-16 h).Finally, the transcription system was added with a DNase I (2 μL of the DNase I was addedper 50 μL of the transcription system), subjected to a water bath at 37°C for 30min to eliminate aplasmid DNA, and the RNA was purified using the RNA purification and concentration kit,quantified with the NanoDrop 2000C, and stored in a refrigerator at -80°C for later use.2. Preparation of a Target DNAFor the target DNA, the first one is that the target DNA was directly added into a Cas12breaction system without amplification. The method was as follows:(1) If the target DNA was a single strand, a 50 bp long oligonucleotide was directly synthesizedas the target DNA (DNMT1-3 (TTC PAM)-R), which contained the 20 bp target sequence(DNMT1-3) recognized by the guide RNA.(2) If the target DNA was double-stranded, two 50 bp long complementary oligonucleotides(DNMT1-3 (TTC PAM)-F; DNMT1-3 (TTC PAM)-R) were directly synthesized, which containedthe 20 bp target sequence (DNMT1-3) recognized by the guide RNA. The two oligonucleotideswere annealed to obtain a short target DNA. Specifically, the paired oligonucleotide (2 μM) wasannealed in a 1X PCR buffer (Transgen Biotech) at a total volume of 20 μL, and then subjected toan annealing procedure: denaturing at 95°C for 5 minutes, and then cooling from 95°C to 20°C ata rate of 1°C per minute using a thermal cycler.(3) the single-stranded or double-stranded target DNA was diluted at a gradient to 2 μM, 0.2μM, 0.02 μM, 0.002 μM and 0.0002 μM for later use.The second one was inserting a fragment containing the target sequence (DNMT1-3) into aplasmid vector for amplification by the LAMP reaction.(1) the fragment containing the target sequence (DNMT1-3) was inserted into a pEasy-BluntZero Cloning Vector using a pEasy-Blunt Zero Cloning Kit from Transgen, so as to obtain thecorrect clone after verified by sequencing.(2) A LAMP expansion reactionUsing the above plasmid as a template, the LAMP amplification reaction was carried out. Thetemplates were added respectively at 0 nM, 1 nM, and 0.1 nM, and diluted at a 10-fold gradient to10-11 nM. The total volume of each reaction system was 25 μL, primers of 1.6 μM of LAMPDNM-FIP and LAMP-DNM-BIP each, 0.2 μM of LAMP-DNM-F3 and LAMP-DNM-B3 each,0.4 μM of LAMP-DNM-LoopF and LAMP-DNM-LoopB each were used, and the kit used for theLAMP reaction was the WarmStart LAMP Kit (NEB). The LAMP reaction procedure was 65°Cfor 30 min. After the LAMP was completed, inactivation was conducted at 80°C for 10 min, andthen the product was directly used for a Cas12b reaction.3. Cas12b reaction(1) annealing of the guide RNA: the guide RNA was diluted to an appropriate concentration (5μM), and annealed in the PCR instrument. The annealing procedure: denaturing at 75°C for 5 min,and then cooling from 75°C to 20°C at a decreasing rate of 1°C per minute.(2) incubation of the guide RNA with Cas12b: the annealed guide RNA was mixed with Cas12bat an equal molar concentration, and allowed to stand at 30°C for 20-30 min.(3) Cas12b reaction: a 20 μL reaction system was added with the mixture of the guide RNAand Cas12b incubated in step (2) (the final concentrations of the guide RNA and Cas12b were both250 μM), 1 μL of a target DNA or 1 μL of a LAMP product, a fluorescence probe (HEX-N12-BHQ1) (with a final concentration of 500 nM), 2 μL of a 10X NEB Buffer 3.1, and 0.5 μL of anRNase inhibitor (40 U / μL). After mixing homogenously, the reaction was conducted at 48°C for30min. After that, it was inactivated fire heating at 98°C for 5 min in a PCR instrument.4. Detection of trans-cleavage activity of Cas12b by a fluorescence microplate reader method:20 μL of the inactivated reaction solution was added into a 96-well plate, and detected by amicroplate reader (with an excitation light at 535 nm and an emission light at 556 nm). Combinedwith the LAMP amplification, Cas12b could produce a significant collateral single-stranded DNAtrans-cleavage activity for a target DNA concentration as low as 10 aM. The results were as shownin FIG. 24.Cis-cleavage characteristics of Cas12a in cleaving a target single-stranded DNA:First, in order to test the single-stranded DNA cleavage characteristics of Cas12a, severalcrRNAs targeting a short single-stranded DNA (DNMT1-3) (Table 1) are designed, which arelabeled with 5(6)-carboxyfluorescein (FAM) at the 3' terminal. After the cleavage by FnCas12a,the reaction product is analyzed by denatured urea-polyacrylamide gel electrophoresis (urea-PAGE). It is found that the single strand DNA cleavage by Cas12a is programmed. That is, thecleavage site is near the 22nd base (21st to 23rd bases) of the target sequence counted from the 3'end base to the 5' end of the first target sequence paired with the crRNA guide sequence, as shownin FIGs. 1A and 1C. The cleavage of a double-stranded DNA by Cas12a requires a PAM sequence,while the cleavage of a single-stranded DNA by Cas12a does not require the PAM sequence (FIGs.1A, 1B and 2), which is similar to the single-stranded DNA cleavage mediated by Cas9. However,the single-stranded DNA cleavage activity mediated by Cas12a depends on a stem-loop structurein crRNA, as shown in FIG. 1A, while Cas9 still shows weak cleavage activity for a singlestrandedDNA with only a 20-nt complementary RNA sequence. The stem-loop structure of thecrRNA is important for stabilizing the structure of Cas12a, which is the reason why the ringstructure of the crRNA is necessary for the cleavage of the single-stranded DNA by Cas12a. It isfurther tested whether a shorter lead sequence crRNA can pass through the single-stranded DNAcleavage site of Cas12a in such a manner that the cleavage is outside the recognition site. Whenthe length of the guide sequence is 16 nt, 18 nt and 20 nt, all of these crRNAs lead to cleavage byCpf1 near the 22nd base, as shown in FIGs. 1B and 1D, meaning that the cleavage site is 4 nt, 2 ntor 0 nt outside the recognition site. Next, the cleavage efficiency of Cas12a on different substratesis tested by using substrates of a double-stranded DNA and a single-stranded DNA respectively,as shown in FIG. 1F. Similar to the situation of Cas9 cleavage, the cleavage of the single-strandedDNA is slower than that of the double-stranded DNA, as shown in FIGs. 1E to 1G. These resultsindicate that the mechanism of recognizing and cleaving a single-stranded DNA by Cas12a maybe different from that of a double-stranded DNA, which is a low-efficiency recognition andcleavage manner independent of PAM; and the PAM sequence accelerates the recognition and / orcleavage of the target double - stranded DNA by C as 1 2 a .Trans-cleavage characteristics of Cas12a in cleaving a single-stranded DNA:When the target single-stranded DNA is labeled at the 3' terminal, Cas12a cleaves at thevicinity of the 22nd base, as shown in FIG. 1. However, when it is labeled at the 5' terminal, nocleavage product band of predicted size is observed, but a short (< 6 nt) FAM-labeled product isproduced, as shown in FIG. 3B. Through detailed experiments, once the ternary complexCas12a / crRNA / target single-stranded DNA is formed, the target single-stranded DNA (DNMT1-3) (Table 1) labeled at 5'-terminal is cleaved, and a short FAM-labeled product is produced, asshown in FIG. 3C. In addition, the ternary complex also cleaves a single-stranded DNA that hasno sequence that is complementary with the crRNA (i.e., a collateral single-stranded DNA) in anyother reaction system, as shown in FIGs. 3C and 3D. This cleavage phenomenon is called transcleavage,which is different from programmable cis-cleavage. When the target single-strandedDNA is labeled at the 3' terminal, trans-cleavage is also observed, but many cis-cleavage productsare left, as shown in FIG. 3B, This may be due to the complex formed by Cas12a / crRNA / targetsingle-stranded DNA, and the target single-stranded DNA is protected, such that its labeled 3'terminal is protected from exposure to a nuclease active site of the ternary complex. These cleavageprocesses can be as shown in FIG . 3 A .In addition to the FnCas12a tested above, 9 Cas12as from other species sources are also tested(Table 2 and FIG. 4A). Except Lb4Cas12a, all Cas12as have good endonuclease activity to theplasmid DNA (as shown in FIG. 4B), and all Cas12a ternary complexes show cis and trans cleavageactivities on the single strand (as shown in FIGs. 4C and 4D). This shows that cis and trans activityof Cas12a on the single-stranded DNA is a common phenomenon.The cis and trans key sites and mechanism for cleaving a single-stranded DNA by Cas12a.In order to determine the key amino acid residues related to cis and trans activities on thesingle-stranded DNA in Cas12a, several candidate residues of Cas12a are mutated to conduct theactivity test. First, three single-amino-acid mutants of FnCas12a (H843A, K852A and K869A) arepurified and tested, and their residues are related to an RNase activity. The results of the study ofthe trans-activity on the single-stranded DNA show that no obvious difference in cis- and transcleavageactivities on the single-stranded DNA between the wild-type FnCas12a and the threemutantsis found , as shown in FIGs . 5 A and 5C .Next, when endonuclease active sites in the FnCas12a, i.e., the RuvC domain (D917A, E1006Aor D1255A) and the Nuc domain (R1218A) sites are mutated, the cis and trans cleavage activitiesof these mutated Cas12a on the single-stranded DNA are both affected, as shown in FIGs. 5B and5D. These results indicate that the key site of Cas12a for cleavage of a target double-strandedDNA is closely related to cis and trans cleavage activities on the single-stranded DNA.Recent structural studies of Cas12b (i.e., C2c1) (including complexes with an extended targetDNA or an extended non-target DNA) show that both strands are located in an RuvC pocket, asshown in FIGs. 6A and 6B. By comparing the endonuclease catalytic residues of Cas12b (i.e.C2c1) and Cas12a, these sites are most likely to play similar roles in the cleavage and functions ofCas12b (i.e., C2c1) and Cas12a. The results of an in vitro single amino acid mutation experimentshow that it is consistent with the above hypothesis. That is, Cas12a is likely to cleave two strandsthrough only one RuvC catalytic pocket .The trans-cleavage activity of a Cas12a complex: in the structure of a complex of Cas12b (i.e.,C2c1) with an additional single-stranded DNA, a sequence-independent single-stranded DNA isalso located on the surface of a catalytic pocket, as shown in FIG. 6C, which is similar to that of acollateral single-stranded DNA substrate in Cas12a. Combined with a single-amino-acid mutationexperiment, it is proposed that the target DNA, non-target DNA and collateral single-strandedDNA are all cleaved in the single RuvC pocket in Cas12a, as shown in FIGs. 6D, 6E and 6F. Theternary Cas12a complex has a collateral single-stranded DNA trans-cleavage activity, while thereason why a monomer or a binary complex does not have the collateral single-stranded DNAtrans-cleavage activity can be explained by comparing the structures of the monomer, binary andternary complexes. The structure of a monomer Cas12a is disordered, the binary complexCas12a / crRNA has a triangular structure, as shown in FIG. 6G, while the ternary complexCas12a / crRNA / target DNA is converted into a double-leaf structure, thus exposing the catalyticpocket to realize trans-cleavage of the collateral single-stranded DNA (as shown in FIG. 6H).Establishment of a nucleic acid detection methodBased on the characteristics of Cas12a, a specific nucleic acid molecule detection method wasdeveloped, which was called HOLMES (one HOur Low-cost Multipurpose Efficient Simpleassay). Just like the name of the technology, it was characterized by a simple test method that isone hour and has low cost , multi pleuse sand high efficiency .In the whole reaction system, the method can be divided into two major steps, one is theamplification of a template nucleic acid, and the other is the specific nucleic acid detection by aCas12a protein. Here, a PCR method is used for nucleic acid amplification, but in fact, anyamplification method can be combined with the nucleic acid detection in the second step, such asthe isothermal amplification method RPA, etc. The initial nucleic acid is not limited to a doublestrandedDNA, but may also be a single-stranded DNA; or even an RNA still can be detected afterreverse transcription, so this method is suitable for various types of nucleic acid molecules. Forthe nucleic acid detection phase, three components are the keys to the experiment, namely Cas12a,crRNA and a nucleic acid probe. In addition to the 10 Cas12as mentioned in the example (these10 proteins are randomly selected), other Cas12a proteins are also suitable for this method. Inaddition, other types of Cas proteins (e.g., a C2c1 protein) are also suitable for the claimed scopeof the present invention: as shown by the experimental results, Alicyclobacillus acidoterrestrisCas12b (i.e., C2c1) also has a collateral single-stranded DNA trans-cleavage activity similar tothat of Cas12a, and its complex with crRNA / target DNA can also cleave a collateral single15s t r a n d e d D N A .For crRNA serving as a guide, it will be more stable in the system after being engineered, forexample being modified manually. With regard to the selection of nucleic acid probes, the presentinvention selects a short single-stranded DNA labeled with HEX and BHQ1, and any otherdetectable labeling method is theoretically applicable, as long as the nucleic acid probe is cleavedto produce detectable differences. Alternatively, the nucleic acid probe can also be designed tofluoresce after binding to a compound, so as to detect whether the probe is cleaved.Furthermore, it should be understood that after reading the above teachings of the presentinvention, those skilled in the art can make various changes or modifications to the presentinvention, and these equivalent forms also fall within the scope defined by the appended claims ofthe present application.Table 1 Substrate sequences of Cas12a characteristic experiment-related cleavageTable 2. Names and GI numbers of the Cas12a protein and the Cas12b (i.e., C2c1) proteinTable 3 Plasmid InformationTable 4 Primers used in the test of the HOLMES methodTable 5 Template sequences for transcription of crRNAPrimers used for detection by DNA amplification through LAMP combined with Cas12a:Table 6 Primers used for amplifying gyrB-1Table 7 Primers used for amplifying gyrB-2Table 8 Primers used for amplifying a rs1467558 siteTable 9 Primers used for amplifying a rs5082 siteTable 10 Primers used for Detection by RPA Amplification combined with Cas12Table 11 Primers used for determining Cas12b having a trans-cleavage activity:Table 12 Primers used for sensitivity test of trans reaction of Cas12bTable 13 Other Sequences Involved in the present inventionAll documents mentioned in the present invention are hereby incorporated by reference as ifeach document is individually incorporated by reference. Furthermore, it should be understood thatafter reading the above teachings of the present invention, those skilled in the art can make variouschanges or modifications to the present invention, and these equivalent forms also fall within thescope defined by the appended claims of the present application.Sequence Listing<110> SHANGHAI TOLO BIOTECHNOLOGY COMPANY LIMITEDSHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES<120> USE OF CAS PROTEIN, METHOD FOR DETECTING TARGET NUCLEIC ACID MOLECULE, AND KIT<130> P2018-0447<150> CN201710573752.0<151> 2017-07-14<160> 187<170> PatentIn version 3.5<210> 1<211> 50<212> DNA<213> Artificial sequence<400> 1aatgtttcct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 2<211> 50<212> DNA<213> Artificial sequence<400> 2gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50<210> 3<211> 50<212> DNA<213> Artificial sequence<400> 3gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50<210> 4<211> 50<212> DNA<213> Artificial sequence<400> 4gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50<210> 5<211> 66<212> DNA<213> Artificial sequence<400> 5tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60ggatcc 66<210> 6<211> 66<212> DNA<213> Artificial sequence<400> 6ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 7<211> 66<212> DNA<213> Artificial sequence<400> 7tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60ggatcc 66<210> 8<211> 66<212> DNA<213> Artificial sequence<400> 8ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 9<211> 66<212> DNA<213> Artificial sequence<400> 9tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60ggatcc 66<210> 10<211> 66<212> DNA<213> Artificial sequence<400> 10ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 11<211> 50<212> DNA<213> Artificial sequence<400> 11gtcacgccac ttgacaggcg agtaacagac atggaccatc aggtttcatt 50<210> 12<211> 50<212> DNA<213> Artificial sequence<400> 12gtcacgccac ttgacaggcg agtaacagac atggaccatc aggccccatt 50<210> 13<211> 50<212> DNA<213> Artificial sequence<400> 13gtcacgccac ttgacaggcg agtaacagac atggaccatc agggggcatt 50<210> 14<211> 50<212> DNA<213> Artificial sequence<400> 14aatgaaacct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 15<211> 50<212> DNA<213> Artificial sequence<400> 15aatggggcct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 16<211> 50<212> DNA<213> Artificial sequence<400> 16aatgccccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 17<211> 11<212> DNA<213> Artificial sequence<400> 17acaaacagaa a 11<210> 18<211> 16<212> DNA<213> Artificial sequence<400> 18cgataacaaa cagaaa 16<210> 19<211> 22<212> DNA<213> Artificial sequence<400> 19aagttgcgat aacaaacaga aa 22<210> 20<211> 28<212> DNA<213> Artificial sequence<400> 20agtagaaagt tgcgataaca aacagaaa 28<210> 21<211> 34<212> DNA<213> Artificial sequence<400> 21gaattcagta gaaagttgcg ataacaaaca gaaa 34<210> 22<211> 24<212> DNA<213> Artificial sequence<400> 22gaattcagta gaaagttgcg ataa 24<210> 23<211> 18<212> DNA<213> Artificial sequence<400> 23agtagaaagt tgcgataa 18<210> 24<211> 12<212> DNA<213> Artificial sequence<400> 24aagttgcgat aa 12<210> 25<211> 12<212> DNA<213> Artificial sequence<220><221> misc_feature<222> (7)..(12)<223> n is a, c, g, or t<400> 25cgataa 12<210> 26<211> 25<212> DNA<213> Artificial sequence<220><221> misc_feature<222> (1)..(25)<223> n is a, c, g, or t<400> 26nnnnnnnnnn nnnnnnnnnn nnnnn 25<210> 27<211> 25<212> DNA<213> Artificial sequence<220><221> misc_feature<222> (1)..(25)<223> n is a, c, g, or t<400> 27nnnnnnnnnn nnnnnnnnnn nnnnn 25<210> 28<211> 66<212> DNA<213> Artificial sequence<400> 28ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 29<211> 28<212> DNA<213> Artificial sequence<400> 29cacaattcca cacaacatac gagccgga 28<210> 30<211> 27<212> DNA<213> Artificial sequence<400> 30tgtagccgta gttaggccac cacttca 27<210> 31<211> 31<212> DNA<213> Artificial sequence<400> 31agttttgtta tcgcaacttt ctactgaatt c 31<210> 32<211> 31<212> DNA<213> Artificial sequence<400> 32agttttgata tcgcaacttt ctactgaatt c 31<210> 33<211> 31<212> DNA<213> Artificial sequence<400> 33agttttgtaa tcgcaacttt ctactgaatt c 31<210> 34<211> 31<212> DNA<213> Artificial sequence<400> 34agttttgttt tcgcaacttt ctactgaatt c 31<210> 35<211> 31<212> DNA<213> Artificial sequence<400> 35agttttgtta acgcaacttt ctactgaatt c 31<210> 36<211> 31<212> DNA<213> Artificial sequence<400> 36agttttgtta tggcaacttt ctactgaatt c 31<210> 37<211> 31<212> DNA<213> Artificial sequence<400> 37agttttgtta tcccaacttt ctactgaatt c 31<210> 38<211> 31<212> DNA<213> Artificial sequence<400> 38agttttgtta tcggaacttt ctactgaatt c 31<210> 39<211> 31<212> DNA<213> Artificial sequence<400> 39agttttgtta tcgctacttt ctactgaatt c 31<210> 40<211> 31<212> DNA<213> Artificial sequence<400> 40agttttgtta tcgcatcttt ctactgaatt c 31<210> 41<211> 31<212> DNA<213> Artificial sequence<400> 41agttttgtta tcgcaagttt ctactgaatt c 31<210> 42<211> 29<212> DNA<213> Artificial sequence<400> 42aaaagttatc gcaactttct actgaattc 29<210> 43<211> 39<212> DNA<213> Artificial sequence<400> 43agttttgtta tcgcaacatt ctactgaatt cggtcatag 39<210> 44<211> 39<212> DNA<213> Artificial sequence<400> 44agttttgtta tcgcaactat ctactgaatt cggtcatag 39<210> 45<211> 39<212> DNA<213> Artificial sequence<400> 45agttttgtta tcgcaactta ctactgaatt cggtcatag 39<210> 46<211> 39<212> DNA<213> Artificial sequence<400> 46agttttgtta tcgcaacttt gtactgaatt cggtcatag 39<210> 47<211> 39<212> DNA<213> Artificial sequence<400> 47agttttgtta tcgcaacttt caactgaatt cggtcatag 39<210> 48<211> 39<212> DNA<213> Artificial sequence<400> 48agttttgtta tcgcaacttt cttctgaatt cggtcatag 39<210> 49<211> 39<212> DNA<213> Artificial sequence<400> 49agttttgtta tcgcaacttt ctagtgaatt cggtcatag 39<210> 50<211> 39<212> DNA<213> Artificial sequence<400> 50agttttgtta tcgcaacttt ctacagaatt cggtcatag 39<210> 51<211> 31<212> DNA<213> Artificial sequence<400> 51agtttagtta tcgcaacttt ctactgaatt c 31<210> 52<211> 31<212> DNA<213> Artificial sequence<400> 52agttatgtta tcgcaacttt ctactgaatt c 31<210> 53<211> 31<212> DNA<213> Artificial sequence<400> 53agtattgtta tcgcaacttt ctactgaatt c 31<210> 54<211> 28<212> DNA<213> Artificial sequence<400> 54agttgtcgtt cctcaactcc ggcgtttc 28<210> 55<211> 28<212> DNA<213> Artificial sequence<400> 55tcgacgccaa taccgtcttt ttcagtgg 28<210> 56<211> 26<212> DNA<213> Artificial sequence<400> 56ctgcctttgc ttctaccttt gcctgt 26<210> 57<211> 25<212> DNA<213> Artificial sequence<400> 57ttgcttctac ctttgcctgt tctgg 25<210> 58<211> 25<212> DNA<213> Artificial sequence<400> 58ttttctggct ggggatggcc gatgg 25<210> 59<211> 50<212> DNA<213> Artificial sequence<400> 59agcaataaca ctaatattga ttccttcaga tatggactcc tttcatagta 50<210> 60<211> 39<212> DNA<213> Artificial sequence<400> 60ttgattcctt cagatatgga ctcctttcat agtataacg 39<210> 61<211> 37<212> DNA<213> Artificial sequence<400> 61tgagcatcgt tattcttacg cgttgtcatt gaaagag 37<210> 62<211> 25<212> DNA<213> Artificial sequence<400> 62agcctgggca acgagtgaaa ctctg 25<210> 63<211> 27<212> DNA<213> Artificial sequence<400> 63acaggaggga caaaggccta agtgtcc 27<210> 64<211> 38<212> DNA<213> Artificial sequence<400> 64catcatagga ttgggaaaag gacatttcag tcattcag 38<210> 65<211> 48<212> DNA<213> Artificial sequence<400> 65agagtccttc tttctcaatt tttcagaata atttagtact ttgggtac 48<210> 66<211> 40<212> DNA<213> Artificial sequence<400> 66cagtactgaa aaaacctgcc tatcaataaa agccctagac 40<210> 67<211> 24<212> DNA<213> Artificial sequence<400> 67gcttcaccgg ctacctttgc tcct 24<210> 68<211> 21<212> DNA<213> Artificial sequence<400> 68ttcacctgca ggccccgcag g 21<210> 69<211> 39<212> DNA<213> Artificial sequence<400> 69cctgactttc aactctgtct ccttcctctt tttacagta 39<210> 70<211> 28<212> DNA<213> Artificial sequence<400> 70tgctgtgact gcttgtagat ggccatgg 28<210> 71<211> 36<212> DNA<213> Artificial sequence<400> 71agtttccaga cctcagtgca caagatactt ttctac 36<210> 72<211> 36<212> DNA<213> Artificial sequence<400> 72acctcagtgc acaagatact tttctacgtc atccac 36<210> 73<211> 33<212> DNA<213> Artificial sequence<400> 73agctccagtg gatggaagat ctttgagatc cag 33<210> 74<211> 40<212> DNA<213> Artificial sequence<400> 74agtcaaagag attcatgcct gggactttaa tcacatttat 40<210> 75<211> 33<212> DNA<213> Artificial sequence<400> 75atgcctggga ctttaatcac atttatcgga agg 33<210> 76<211> 31<212> DNA<213> Artificial sequence<400> 76caaatctgtc tccacctctc agctcacctt g 31<210> 77<211> 34<212> DNA<213> Artificial sequence<400> 77ttcttgaacc caaactcacc tggcatttaa actg 34<210> 78<211> 33<212> DNA<213> Artificial sequence<400> 78aaactcacct ggcatttaaa ctgactctgt aag 33<210> 79<211> 33<212> DNA<213> Artificial sequence<400> 79aaactcacct ggcatttaaa ctgtctctgt aag 33<210> 80<211> 27<212> DNA<213> Artificial sequence<400> 80tgccgaggct gagttcagct actctcc 27<210> 81<211> 26<212> DNA<213> Artificial sequence<400> 81acacagcacc gtgggtcaga ccttgc 26<210> 82<211> 26<212> DNA<213> Artificial sequence<400> 82tgggtcagac tttgccggtg agagtc 26<210> 83<211> 26<212> DNA<213> Artificial sequence<400> 83tgggtcagac tttgctggtg agagtc 26<210> 84<211> 27<212> DNA<213> Artificial sequence<400> 84agcagtggcc atgtgatgct gatgatg 27<210> 85<211> 27<212> DNA<213> Artificial sequence<400> 85ccccggctct gttggctttg agaattg 27<210> 86<211> 33<212> DNA<213> Artificial sequence<400> 86ctctgttggc tttgagaatt gcctgtctgt gtc 33<210> 87<211> 33<212> DNA<213> Artificial sequence<400> 87ctctgttggc tttgagaatt gtctgtctgt gtc 33<210> 88<211> 25<212> DNA<213> Artificial sequence<400> 88accgatacct ggcagccctt ggatg 25<210> 89<211> 12<212> DNA<213> Artificial sequence<220><221> misc_feature<222> (1)..(12)<223> n is a, c, g, or tPage 10P20180447-seql_app_English<400> 89nnnnnnnnnn nn 12<210> 90<211> 25<212> DNA<213> Artificial sequence<400> 90gaaattaata cgactcacta taggg 25<210> 91<211> 68<212> DNA<213> Artificial sequence<400> 91gaattcagta gaaagttgcg ataaatctac aacagtagaa attccctata gtgagtcgta 60ttaatttc 68<210> 92<211> 59<212> DNA<213> Artificial sequence<400> 92agaaagttgc gataaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59<210> 93<211> 60<212> DNA<213> Artificial sequence<400> 93tagaaagttg cgataaatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 94<211> 61<212> DNA<213> Artificial sequence<400> 94gtagaaagtt gcgataaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 95<211> 62<212> DNA<213> Artificial sequence<400> 95agtagaaagt tgcgataaat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60tc 62<210> 96<211> 67<212> DNA<213> Artificial sequence<400> 96gagtaacaga catggaccat cagatctaca acagtagaaa ttccctatag tgagtcgtat 60taatttc 67<210> 97<211> 67<212> DNA<213> Artificial sequence<400> 97gacatggacc atcaggaaac attatctaca acagtagaaa ttccctatag tgagtcgtat 60taatttc 67<210> 98<211> 67<212> DNA<213> Artificial sequence<400> 98aggcgagtaa cagacatgga ccaatctaca acagtagaaa ttccctatag tgagtcgtat 60taatttc 67<210> 99<211> 67<212> DNA<213> Artificial sequence<400> 99tgacaggcga gtaacagaca tggatctaca acagtagaaa ttccctatag tgagtcgtat 60taatttc 67<210> 100<211> 60<212> DNA<213> Artificial sequence<400> 100agacatggac catcagatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 101<211> 62<212> DNA<213> Artificial sequence<400> 101acagacatgg accatcagat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60tc 62<210> 102<211> 64<212> DNA<213> Artificial sequence<400> 102taacagacat ggaccatcag atctacaaca gtagaaattc cctatagtga gtcgtattaa 60tttc 64<210> 103<211> 48<212> DNA<213> Artificial sequence<400> 103gacatggacc atcaggaaac attccctata gtgagtcgta ttaatttc 48<210> 104<211> 48<212> DNA<213> Artificial sequence<400> 104aggcgagtaa cagacatgga ccaccctata gtgagtcgta ttaatttc 48<210> 105<211> 48<212> DNA<213> Artificial sequence<400> 105tgacaggcga gtaacagaca tggccctata gtgagtcgta ttaatttc 48<210> 106<211> 61<212> DNA<213> Artificial sequence<400> 106cctcttccca gaacaggatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 107<211> 61<212> DNA<213> Artificial sequence<400> 107cctcttccca gcacaggatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 108<211> 61<212> DNA<213> Artificial sequence<400> 108ctgaagcgtt atactatatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 109<211> 61<212> DNA<213> Artificial sequence<400> 109ctgaagcgtt gtactatatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 110<211> 60<212> DNA<213> Artificial sequence<400> 110ttttatctga atgattatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 111<211> 60<212> DNA<213> Artificial sequence<400> 111ttttatctga atgactatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 112<211> 61<212> DNA<213> Artificial sequence<400> 112aaaaaagagt gagtaccatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 113<211> 61<212> DNA<213> Artificial sequence<400> 113aaaaaagagt gggtaccatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 114<211> 61<212> DNA<213> Artificial sequence<400> 114ggtagaaggt ccaggagatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 115<211> 61<212> DNA<213> Artificial sequence<400> 115ggtagaaggt ctaggagatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 116<211> 60<212> DNA<213> Artificial sequence<400> 116gggcagggga gtactgatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 117<211> 60<212> DNA<213> Artificial sequence<400> 117gggcagggga ctactgatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 118<211> 59<212> DNA<213> Artificial sequence<400> 118tcagtggatg atgtaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59<210> 119<211> 59<212> DNA<213> Artificial sequence<400> 119tcagtggatg acgtaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59<210> 120<211> 61<212> DNA<213> Artificial sequence<400> 120ggaaattctc cttccgaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 121<211> 61<212> DNA<213> Artificial sequence<400> 121ggaaattctc cttccaaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 122<211> 60<212> DNA<213> Artificial sequence<400> 122tcttacagag tcagttatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 123<211> 60<212> DNA<213> Artificial sequence<400> 123tcttacagag ccagttatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60<210> 124<211> 61<212> DNA<213> Artificial sequence<400> 124gtcttacaga gacagttatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 125<211> 59<212> DNA<213> Artificial sequence<400> 125ctggactctc accggatcta caacagtaga aattccctat agtgagtcgt attaatttc 59<210> 126<211> 59<212> DNA<213> Artificial sequence<400> 126ctggactctc accagatcta caacagtaga aattccctat agtgagtcgt attaatttc 59<210> 127<211> 61<212> DNA<213> Artificial sequence<400> 127cacagacagg caattctatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 128<211> 61<212> DNA<213> Artificial sequence<400> 128cacagacaga caattctatc tacaacagta gaaattccct atagtgagtc gtattaattt 60c 61<210> 129<211> 68<212> DNA<213> Artificial sequence<400> 129tcgcgcttgt cgcgcagacg aatgatctac aacagtagaa attccctata gtgagtcgta 60ttaatttc 68<210> 130<211> 18<212> DNA<213> Artificial sequence<400> 130catggtgcgt ttctggcc 18<210> 131<211> 20<212> DNA<213> Artificial sequencePage 15P20180447-seql_app_English<400> 131cggcgttttg ttcttgttca 20<210> 132<211> 42<212> DNA<213> Artificial sequence<400> 132acaactcacg cagacgtttc gcaaccttca ccaatgtgac cg 42<210> 133<211> 41<212> DNA<213> Artificial sequence<400> 133gttcctcaac tccggcgttt ccgatgccgc cttcatagtg g 41<210> 134<211> 20<212> DNA<213> Artificial sequence<400> 134cagaatttca tattcgaact 20<210> 135<211> 20<212> DNA<213> Artificial sequence<400> 135gacggcaaag aagaccactt 20<210> 136<211> 18<212> DNA<213> Artificial sequence<400> 136cgacggcaaa gaagacca 18<210> 137<211> 18<212> DNA<213> Artificial sequence<400> 137agcctgccag gtgagtac 18<210> 138<211> 40<212> DNA<213> Artificial sequence<400> 138cgggtggatc ggcgttttgt tcactatgaa ggcggcatca 40<210> 139<211> 40<212> DNA<213> Artificial sequence<400> 139gtattggcgt cgaagtggcg ttcgctgcgg aatgttgttg 40<210> 140<211> 22<212> DNA<213> Artificial sequence<400> 140ttgttcagat attcaacgaa cg 22<210> 141<211> 20<212> DNA<213> Artificial sequence<400> 141gtggaacgat ggcttccagg 20<210> 142<211> 19<212> DNA<213> Artificial sequence<400> 142cagctgtaga ccataagcc 19<210> 143<211> 18<212> DNA<213> Artificial sequence<400> 143gtggctgagc atcgttat 18<210> 144<211> 48<212> DNA<213> Artificial sequence<400> 144actatgaaag gagtccatat ctgaaggaat tcaggtagtg gtttggga 48<210> 145<211> 40<212> DNA<213> Artificial sequence<400> 145gcttcagcct actgcaaatc ctacgcgttg tcattgaaag 40<210> 146<211> 22<212> DNA<213> Artificial sequence<400> 146tcaatattag tgttattgct tg 22<210> 147<211> 22<212> DNA<213> Artificial sequence<400> 147tggtggaaga tttggacagg ac 22<210> 148<211> 19<212> DNA<213> Artificial sequence<400> 148gctggaaagg tcaagggac 19<210> 149<211> 19<212> DNA<213> Artificial sequence<400> 149ggggtttgtt gcacagtcc 19<210> 150<211> 44<212> DNA<213> Artificial sequence<400> 150caaaggtaga agcaaaggca ggaggtttgc ccaaggtcac acag 44<210> 151<211> 41<212> DNA<213> Artificial sequence<400> 151ctgggaagag ggagggctca gtgttgccac actttcactg g 41<210> 152<211> 18<212> DNA<213> Artificial sequence<400> 152gtgagcgggt ggggtgct 18<210> 153<211> 23<212> DNA<213> Artificial sequence<400> 153tctaagtctt ccagcacggg atc 23<210> 154<211> 34<212> DNA<213> Artificial sequence<400> 154atatgaaatt ctggcgaaac gtctgcgtga gttg 34<210> 155<211> 33<212> DNA<213> Artificial sequence<400> 155aaacgtctgc gtgagttgtc gttcctcaac tcc 33<210> 156<211> 33<212> DNA<213> Artificial sequence<400> 156acttcgacgc caataccgtc tttttcagtg gag 33<210> 157<211> 59<212> DNA<213> Artificial sequence<400> 157atctgagaag tggcacttat cgcaactttc tactgaggtc atagctgttt cctgtgtga 59<210> 158<211> 59<212> DNA<213> Artificial sequence<400> 158gtcctctaga cccctatagt gagtcgtatt aatttcatga ttacgaattc gagctcggt 59<210> 159<211> 58<212> DNA<213> Artificial sequence<400> 159ccactttcca ggtggcaaag cccgttgagc ttctcaaatc tgagaagtgg cacttatc 58<210> 160<211> 59<212> DNA<213> Artificial sequence<400> 160tggaaagtgg ccattggcac acccgttgaa aaattctgtc ctctagaccc ctatagtga 59<210> 161<211> 25<212> DNA<213> Artificial sequence<400> 161gaaattaata cgactcacta taggg 25<210> 162<211> 24<212> DNA<213> Artificial sequence<400> 162tcagtagaaa gttgcgataa gtgc 24<210> 163<211> 23<212> DNA<213> Artificial sequence<400> 163aacagacatg gaccatcagg gtg 23<210> 164<211> 66<212> DNA<213> Artificial sequence<400> 164tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60ggatcc 66<210> 165<211> 66<212> DNA<213> Artificial sequence<400> 165ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 166<211> 50<212> DNA<213> Artificial sequence<400> 166gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50<210> 167<211> 66<212> DNA<213> Artificial sequence<400> 167ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60cagaaa 66<210> 168<211> 66<212> DNA<213> Artificial sequence<400> 168tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60ggatcc 66<210> 169<211> 42<212> DNA<213> Artificial sequence<400> 169cctgatggtc catgtctgtt ggtcatagct gtttcctgtg tg 42<210> 170<211> 36<212> DNA<213> Artificial sequence<400> 170tggaccatca gggtgccact tctcagattt gagaag 36<210> 171<211> 25<212> DNA<213> Artificial sequence<400> 171gaaattaata cgactcacta taggg 25<210> 172<211> 23<212> DNA<213> Artificial sequence<400> 172aacagacatg gaccatcagg gtg 23<210> 173<211> 50<212> DNA<213> Artificial sequence<400> 173aatgttccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 174<211> 50<212> DNA<213> Artificial sequence<400> 174gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50<210> 175<211> 20<212> DNA<213> Artificial sequence<400> 175gtgaacgttc ccttagcact 20<210> 176<211> 19<212> DNA<213> Artificial sequence<400> 176gggagggcag aactagtcc 19<210> 177<211> 41<212> DNA<213> Artificial sequence<400> 177cgccacttga caggcgagta actgccactt attgggtcag c 41<210> 178<211> 39<212> DNA<213> Artificial sequence<400> 178gcgtgttccc cagagtgact tagcagcttc ctcctcctt 39<210> 179<211> 22<212> DNA<213> Artificial sequence<400> 179aggaaacatt aacgtactga tg 22<210> 180<211> 21<212> DNA<213> Artificial sequence<400> 180ttccttttat ttcccttcag c 21<210> 181<211> 50<212> DNA<213> Artificial sequence<400> 181gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50<210> 182<211> 50<212> DNA<213> Artificial sequence<400> 182aatgttccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50<210> 183<211> 111<212> DNA<213> Artificial sequence<400> 183gtctagagga cagaattttt caacgggtgt gccaatggcc actttccagg tggcaaagcc 60cgttgagctt ctcaaatctg agaagtggca ccctgatggt ccatgtctgt t 111<210> 184<211> 20<212> DNA<213> Artificial sequence<400> 184cctgatggtc catgtctgtt 20<210> 185<211> 50<212> DNA<213> Artificial sequence<400> 185gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50<210> 186<211> 50<212> DNA<213> Artificial sequence<400> 186gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50<210> 187<211> 1129<212> PRT<213> Alicyclobacillus acidoterrestris<400> 187Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro1 5 10 15Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly20 25 30Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu35 40 45Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr50 55 60Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln65 70 75 80Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu85 90 95Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly100 105 110Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu115 120 125Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn130 135 140Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu145 150 155 160Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala165 170 175Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg180 185 190Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg195 200 205Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala210 215 220Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln225 230 235 240Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn245 250 255Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln260 265 270Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala275 280 285His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu290 295 300Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala305 310 315 320Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His325 330 335Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg340 345 350Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu355 360 365Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp370 375 380Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn385 390 395 400Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His405 410 415Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg420 425 430Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp435 440 445Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg450 455 460Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys465 470 475 480Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly485 490 495Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser500 505 510Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu515 520 525Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp530 535 540Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu545 550 555 560Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser565 570 575Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro580 585 590Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp595 600 605Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly610 615 620Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg625 630 635 640Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val645 650 655Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys660 665 670Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp675 680 685Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly690 695 700Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg705 710 715 720Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp725 730 735Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val740 745 750Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr755 760 765Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val770 775 780Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His785 790 795 800Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile805 810 815Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys820 825 830Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu835 840 845Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn850 855 860Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile865 870 875 880Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala885 890 895Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys900 905 910Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe915 920 925Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys930 935 940Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe945 950 955 960Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala965 970 975Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe980 985 990Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly995 1000 1005Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp1010 1015 1020Ser Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr1025 1030 1035Tyr Glu Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln1040 1045 1050Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp1055 1060 1065Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly1070 1075 1080Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser1085 1090 1095Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln Ile Arg1100 1105 1110Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly Asp1115 1120 1125Ile

Claims

1. A method for detecting a target nucleic acid molecule, comprising adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer solution into a system comprising the target nucleic acid molecule to be detected, and then detecting the nucleic acid probe.

2. The method for detecting a target nucleic acid molecule according to claim 1, wherein the Cas protein is Cas12a or a Cas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a; the Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; and the Cas12a is preferably LbCas12a.

3. The method for detecting a target nucleic acid molecule according to claim 1, wherein the guide RNA refers to an RNA that guides the Cas protein to specifically bind to a target DNA.

4. The method for detecting a target nucleic acid molecule according to claim 1, wherein 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 fluorescent group HEX at a 5' terminal and labeled with a quenching group BHQ1 at a 3' terminal; preferably, the method for detecting the nucleic acid probe is preferably a fluorescence detection method; and the fluorescence detection method is preferably a detection method using a microplate reader or a fluorescence spectrophotometer.

5. The method for detecting a target nucleic acid molecule according to any one of claims 1 to 4, wherein the target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained by amplification.

6. The method for detecting a target nucleic acid molecule according to claim 5, wherein the detection method can detect a pathogenic microorganism, gene mutation, or a specific target DNA.

7. The method according to claim 1, wherein the Cas protein comprises Cas12b (i.e., C2c1).

8. Use of a Cas protein in a method for detecting a target nucleic acid molecule.

9. The use according to claim 8, wherein when a target DNA, a guide RNA and a Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system; and preferably, the guide RNA refers to an RNA that guides the Cas protein to specifically bind to the target DNA.

10. A kit for detecting a target nucleic acid molecule, comprising a guide RNA, a Cas protein and a nucleic acid probe.

11. A detection system for detecting a target nucleic acid molecule, comprising: (a) a Cas protein, which is Cas12a or a Cas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a; (b) a guide RNA that guides the Cas protein to specifically bind to the target nucleic acid molecule; and (c) a nucleic acid probe which is a single-stranded DNA; wherein the target nucleic acid molecule is a target DNA.

12. The detection system according to claim 11, wherein the Cas protein having a collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from a group consisting of Cas12b (i.e., C2c1).

13. The detection system according to claim 11, wherein the nucleic acid probe comprises a single-stranded DNA carrying a detectable label.

14. A kit for detecting a target nucleic acid molecule, comprising: i) a first container and a Cas protein in the first container, the Cas protein being Cas12a or Cas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a; ii) an optional second container and a guide RNA in the second container, the guide RNA guiding the Cas protein to specifically bind to the target nucleic acid molecule; iii) a third container and a nucleic acid probe in the third container; iv) an optional fourth container and a buffer solution in the fourth container; wherein the target nucleic acid molecule is a target DNA.

15. A method for detecting whether a target nucleic acid molecule exists in a sample, comprising the following steps: (a) providing the detection system for detecting a target nucleic acid molecule according to claim 11, wherein the detection system further comprises a sample to be detected; and (b) detecting whether the nucleic acid probe in the detection system is cleaved by a Cas protein, wherein the cleavage is a trans-cleavage of a collateral single-stranded DNA; wherein if the nucleic acid probe is cleaved by the Cas protein, then it 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, then it indicates the absence of the target nucleic acid molecule in the sample.

16. The method according to claim 15, wherein a method for amplifying the nucleic acid is selected from a group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2.

17. The method according to claim 15, wherein when the upstream and downstream (in a range of -20 nt to +20 nt, preferably in a range of -15 nt to +15 nt, and more preferably in a range of -10 nt to +10 nt) of a target site lack a PAM sequence, nucleic acid amplification is carried out using a PAM-introducing primer.

18. The method according to claim 17, wherein the PAM-introducing 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' terminal and is complementary or noncomplementary to the sequence of the target nucleic acid molecule; P2 is a PAM sequence; and P3 is a 3' segment sequence that is located at the 3' terminal and is complementary to the sequence of the target nucleic acid molecule.

19. The method according to claim 15, wherein when the upstream and downstream (in a range of -20 nt to +20 nt, preferably in a range of -15 nt to +15 nt, and more preferably in a 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 contains the PAM sequence.

20. Use of a Cas protein in preparation of a detection reagent or a kit for detecting a target nucleic acid molecule based on collateral single-stranded DNA cleavage, wherein the Cas protein is Cas12a or a Cas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a.

21. The use according to claim 20, wherein the Cas protein having a collateral single-stranded DNA cleavage activity similar to that of the Cas12a is selected from a group consisting of Cas12b (or C2c1).