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WO2019011022A1 - 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒 - Google Patents

一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒 Download PDF

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WO2019011022A1
WO2019011022A1 PCT/CN2018/082769 CN2018082769W WO2019011022A1 WO 2019011022 A1 WO2019011022 A1 WO 2019011022A1 CN 2018082769 W CN2018082769 W CN 2018082769W WO 2019011022 A1 WO2019011022 A1 WO 2019011022A1
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Prior art keywords
nucleic acid
target
cas12a
acid molecule
cas protein
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PCT/CN2018/082769
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English (en)
French (fr)
Inventor
成秋香
李诗渊
李晓晏
李林显
王金
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Shanghai Tolo Biotechnology Co Ltd
Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Tolo Biotechnology Co Ltd
Shanghai Institutes for Biological Sciences SIBS of CAS
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Priority to NZ760987A priority Critical patent/NZ760987B2/en
Priority to BR112020000809-5A priority patent/BR112020000809A2/pt
Priority to MX2020000481A priority patent/MX2020000481A/es
Priority to EA202090290A priority patent/EA202090290A1/ru
Priority to CN201880046701.5A priority patent/CN111094588B/zh
Priority to CN202410308071.1A priority patent/CN118207297A/zh
Priority to KR1020207003220A priority patent/KR102822761B1/ko
Priority to EP18832456.0A priority patent/EP3653722A4/en
Priority to MYPI2020000224A priority patent/MY209429A/en
Priority to AU2018299445A priority patent/AU2018299445B2/en
Priority to IL272005A priority patent/IL272005B1/en
Priority to SG11202000336RA priority patent/SG11202000336RA/en
Application filed by Shanghai Tolo Biotechnology Co Ltd, Shanghai Institutes for Biological Sciences SIBS of CAS filed Critical Shanghai Tolo Biotechnology Co Ltd
Priority to CA3069788A priority patent/CA3069788A1/en
Priority to JP2020523474A priority patent/JP7778338B2/ja
Priority to US16/631,157 priority patent/US12180539B2/en
Publication of WO2019011022A1 publication Critical patent/WO2019011022A1/zh
Priority to PH12020500103A priority patent/PH12020500103A1/en
Anticipated expiration legal-status Critical
Priority to ZA2020/00682A priority patent/ZA202000682B/en
Priority to US17/224,541 priority patent/US11584955B2/en
Priority to US18/085,121 priority patent/US20230131421A1/en
Priority to JP2023065743A priority patent/JP2023080282A/ja
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is in the field of biotechnology, and in particular, the invention relates to a method for the detection of target nucleic acid molecules.
  • Nucleic acid detection methods have important application value, such as detection of pathogens and detection of genetic diseases.
  • nucleic acid diagnostics NADs
  • Microbial contamination detection human pathogen infection and other fields are of great significance.
  • Another aspect is the detection of single nucleotide polymorphisms (SNPs) in humans or other species. Understanding the relationship between genetic variation and biological function at the genomic level provides a new perspective for modern molecular biology, in which SNPs are closely related to biological functions, evolution, and disease, so the development of SNPs detection and analysis techniques Particularly important.
  • RNA molecules are very stable, so the test sample can be derived from a series of complex biological samples; RNA is very susceptible to degradation, so care must be taken when handling it.
  • restriction endonuclease digestion assays were established, and Southern, Northern, and dot blot hybridization methods were developed for specific detection of nucleic acid molecules.
  • PCR method became a routine experimental method, it led to an exponential progress in molecular biology.
  • PCR technology is the first and most commonly used amplification method. Based on the PCR method, fluorescent labeling probes are introduced to detect the amplification of targets in real time, called Realtime PCR. Realtime PCR is not only a fast and sensitive method, but it can also be used for quantitative analysis.
  • ligase chain reaction In addition to PCR amplification methods, many alternative methods have been established, such as ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, Loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA) and Recombinase Polymerase. Amplification (RPA) and so on.
  • LAMP Loop-mediated isothermal amplification
  • RCA rolling circle amplification
  • RPA Recombinase Polymerase.
  • the advantage of many of these alternatives is isothermal, which means that only one temperature is required to complete the reaction without the need for a thermal cycling instrument like PCR.
  • Methods for nucleic acid detection In addition to Realtime PCR, which can directly perform amplification and detection, FISH hybrid in situ hybridization is the most commonly used detection method - in situ hybridization with complementary target sequences by labeling molecular probes. In addition, detection methods such as next-generation sequencing technologies and Oxford Nanopore sequencing technologies have been developed
  • the detection of SNPs first requires amplification of a method such as PCR to obtain a sufficient amount of SNP-containing region fragments for further detection.
  • a method such as PCR to obtain a sufficient amount of SNP-containing region fragments for further detection.
  • the more common methods are: primer extension, hybridization, ligation and enzymatic cleavage.
  • a specific method such as mass spectrometry, fluorescence detection, chemiluminescence detection, and the like.
  • bypass activity means that Cas13a binds to a specific target RNA and then cleaves other non-target RNAs randomly (here, the RNA molecule is designed as an RNA fluorescence reporter system); by combining with isothermal amplification technology RPA to achieve rapid target RNA detection;
  • the Zhang Feng team called this detection method SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).
  • SHERLOCK method involves the binding of an RNA template, so if it is necessary to detect DNA, it is necessary to first transcribe the DNA into an RNA template for detection; in view of the instability of the RNA, the method will undoubtedly increase the difficulty of the operation.
  • Cas12a is an RNA-directed specific DNA endonuclease like the commonly used Cas9 protein; Cas12a has its own characteristics, such as the need for crRNA only to guide the specific cleavage of double-stranded DNA, and the production of sticky ends.
  • Another object of the present invention is to provide a use of a Cas protein in a method for detecting a target nucleic acid molecule.
  • a kit comprising a guide RNA, a Cas protein, a nucleic acid probe, a buffer.
  • a method for detecting a target nucleic acid molecule which comprises adding a guide RNA, a Cas protein, a nucleic acid probe, a buffer solution, and then detecting the reaction system containing the target nucleic acid molecule to be detected (especially by detecting the fluorescence intensity) Detection).
  • the Cas protein is Cas12a or has a Cas protein similar to the bypass single-strand DNA cleavage activity of Cas12a.
  • the Cas protein is Cas12a.
  • the Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.
  • the Cas12a is LbCas12a.
  • the guide RNA refers to an RNA that directs the Cas protein to specifically bind to the target DNA.
  • the nucleic acid probe is single-stranded DNA; the single-stranded DNA is preferably a fluorescently-labeled single-stranded DNA; and the single-stranded DNA is preferably labeled with a fluorescent group HEX at the 5' end and A fluorescent probe after labeling the quenching group BHQ1 at the 3' end.
  • the detection method of the nucleic acid probe is preferably a fluorescence detection method; and the fluorescence detection method is preferably a method of detecting using a microplate reader or a fluorescence spectrophotometer.
  • the target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained after amplification.
  • the detection method of the present invention detects pathogenic microorganisms, gene mutations or specific target DNA
  • the Cas protein comprises Cas12b (C2c1).
  • a Cas protein for the detection of a target nucleic acid molecule, or for the preparation of a preparation for the detection of a target nucleic acid molecule.
  • the complex cleaves other single-stranded DNA molecules in the system.
  • the guide RNA refers to an RNA that directs the Cas protein to specifically bind to the target DNA.
  • a kit comprising a guide RNA, a Cas protein, a nucleic acid probe.
  • the kit further comprises a buffer.
  • a detection system for detecting a target nucleic acid molecule comprising:
  • target nucleic acid molecule is a target DNA.
  • the detection system further comprises (d) a buffer.
  • the detection system further comprises a target nucleic acid molecule to be detected.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1-100 copies/ ⁇ l or 10 15 copies/ ⁇ L, preferably 1-10 copies/ ⁇ L. More preferably 1-5 copies / microliters.
  • the molar ratio of the nucleic acid probe to the target nucleic acid molecule is from 10 3 :1 to 10 14 :1 , preferably 10 4 :1 to 10 7 : 1.
  • the detection site of the target nucleic acid molecule is located at positions 1-12 downstream of the PAM sequence of the guide RNA.
  • the guide RNA is 15-30 nt in length, preferably 15-18 nt.
  • the target DNA comprises cDNA.
  • the target DNA is selected from the group consisting of single-stranded DNA, double-stranded DNA, or a combination thereof.
  • the nucleic acid probe has a fluorescent group and a quenching group.
  • the fluorophore and the quenching group are each independently located at the 5' end, the 3' end, and the middle of the nucleic acid probe.
  • the nucleic acid probe has a length of from 3 to 300 nt, preferably from 5 to 100 nt, more preferably from 6 to 50 nt, most preferably from 8 to 20 nt.
  • the target nucleic acid molecule comprises a target nucleic acid molecule derived from a group selected from the group consisting of a plant, an animal, an insect, a microorganism, a virus, or a combination thereof.
  • the target DNA is synthetic or naturally occurring DNA.
  • the target DNA comprises wild-type or mutant DNA.
  • the target DNA includes DNA obtained by reverse transcription or amplification of RNA, such as cDNA and the like.
  • the Cas12a is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; more preferably, the Cas12a is LbCas12a.
  • the Cas protein having a bypass single-strand DNA cleavage activity similar to Cas12a is selected from the group consisting of Cas12b (i.e., C2c1).
  • the Cas12b protein is selected from the group consisting of AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus), AkCas12b (Alicyclobacillus kakegawensis), AmCas12b (Alicyclobacillus macrosporangiidus), AhCas12b (Alicyclobacillus herbarius), AcCas12b (Alicyclobacillus) Contaminans).
  • AacCas12b Alicyclobacillus acidoterrestris
  • Aac2Cas12b Alicyclobacillus acidiphilus
  • AkCas12b Alicyclobacillus kakegawensis
  • AmCas12b Alicyclobacillus macrosporangiidus
  • AhCas12b Alicyclobacillus herbarius
  • the nucleic acid probe comprises a single-stranded DNA with a detectable label.
  • the single-stranded DNA is fluorescent and biotinylated single-stranded DNA.
  • the single-stranded DNA is a fluorescently labeled single-stranded DNA.
  • the single-stranded DNA is a fluorescent probe after the fluorescent group HEX is labeled at the 5' end and the quenching group BHQ1 is labeled at the 3' end.
  • a kit for detecting a target nucleic acid molecule comprising:
  • a first container and a Cas protein located in the first container the Cas protein being Cas12a or having a Cas protein similar to the bypass single-strand DNA cleavage activity of Cas12a;
  • target nucleic acid molecule is a target DNA.
  • any two, three, or four (or all) of the first, second, third, and fourth containers may be the same or different containers.
  • the nucleic acid probe has a fluorescent group and a quenching group.
  • a method for detecting the presence or absence of a target nucleic acid molecule in a sample comprising the steps of:
  • nucleic acid probe is cleaved by the Cas protein, indicating that the target nucleic acid molecule is present in the sample; and the nucleic acid probe is not cleaved by the Cas protein, indicating that the target nucleic acid molecule is not present in the sample.
  • the sample to be detected includes an unamplified sample and an amplified (or nucleic acid amplified) sample.
  • the sample to be detected is a sample obtained by amplification.
  • the nucleic acid amplification method is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification. Increase, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
  • the PCR comprises high temperature PCR, normal temperature PCR, and low temperature PCR.
  • the method is for detecting whether a nucleic acid at a target site is at a SNP, a point mutation, a deletion, and/or an insertion.
  • nucleic acid amplification is carried out using primers introduced with PAM.
  • the primer introduced into the PAM has a structure of formula I from 5' to 3':
  • P1 is a 5' segment sequence at the 5' end that is complementary or non-complementary to the sequence of the target nucleic acid molecule;
  • P2 is a PAM sequence
  • P3 is a 3' segment sequence complementary to the sequence of the target nucleic acid molecule at the 3' end.
  • the PAM primer specifically binds upstream or downstream of the target nucleic acid molecule.
  • P1 has a length of 0-20 nt.
  • P3 has a length of 5-20 nt.
  • the PAM primer has a length of 18 to 50 nt, preferably 20 to 35 nt.
  • the complementation comprises fully complementary and partially complementary.
  • At least one primer is used in the nucleic acid amplification to contain a PAM sequence.
  • PAM is contained in the upstream and downstream of the target site (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, more preferably in the range of -10 nt to +10 nt).
  • primers with or without a PAM sequence may be employed, and the amplified amplification product contains the PAM sequence.
  • the detection in step (b) comprises a fluorescence detection method.
  • the fluorescence detection method is performed using a microplate reader or a fluorescence spectrophotometer.
  • a Cas protein for the preparation of a detection reagent or kit for detecting a target nucleic acid molecule based on a bypass single-stranded DNA cleavage, wherein the Cas protein is Cas12a or has The bypass single-stranded DNA cleavage of Cas12a is similar to the Cas protein.
  • the Cas12a is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combination thereof; more preferably, the Cas12a is LbCas12a.
  • the Cas protein having a bypass single-strand DNA cleavage activity similar to Cas12a is selected from the group consisting of Cas12b (or C2c1).
  • the Cas12b protein is selected from the group consisting of AacCas12b.
  • Figure 1 shows the cis-cut (cis-cut) properties of Cas12a cleavage target single-stranded DNA.
  • Figure 2 shows the PAM sequence required for Cas12a to cleave target single-stranded DNA independent of cleavage of the double strand.
  • Figure 3 shows the trans-cut (trans-cut) properties of Cas12a cleavage of single-stranded DNA.
  • Figure 4 shows Cas12a from 10 different sources tested, all of which have cis-cut and trans-cleavage activities of single-stranded DNA.
  • Figure 5 identifies sites in Cas12a that may be associated with cis-cleavage and trans-cleavage activity of single-stranded DNA by single point mutation experiments of Cas12a.
  • Figure 6 shows the structure of Cas12a and Cas12b (i.e., C2c1) monomers and their complexes with guide RNA and target DNA.
  • Figure 7 shows the fluorescence values obtained for different Cas12a using a specific double-stranded DNA substrate using single-stranded DNA (HEX-N12-BHQ1) as a fluorescent detection probe.
  • the negative control group did not add a specific substrate.
  • Figure 8 shows a schematic flow diagram of the HOLMES method for detecting target DNA based on target DNA amplification and Cas12a trans-cleavage activity against the single-stranded DNA.
  • Figure 9 shows the sensitivity test of target DNA directly using FnCas12a or LbCas12a, or in combination with the HOLMES method.
  • Figure 10 shows the HOLMES fluorescence detection values of the target sequences of different unit point mutations of the crRNA binding FnCas12a or LbCas12a of different lengths of the guide sequence.
  • Figure 11 is a FAM-labeled fluorescent probe using 10 Cas12a proteins to test whether a FAM-labeled single-stranded DNA probe is trans-cut after addition of target single-stranded DNA.
  • Figure 12 uses HEX-N12-BHQ1 as a probe and tested for fluorescence values after 10 Cas12a proteins were tested for addition to target single-stranded DNA.
  • Fig. 13(A) shows the HOLMES detection value of the pure culture Escherichia coli MG1655 as a positive control template with the gyrB gene fragment as the target sequence using HEX and BHQ1 for single-stranded DNA fluorescent probes. It is shown that as the concentration of E. coli MG1655 decreases, its fluorescence response value gradually decreases.
  • B Detected values of water samples in different locations.
  • Figure 14 shows the flow diagram of the HOLMES method for detecting SNPs, and the fluorescence detection values of the five SNP sites.
  • Figure 15 shows the HOLMES method for detecting fluorescence detection values of key sites in the TP53 gene (cancer-associated gene).
  • Figure 16 shows the detection of the detection of 5 SNP loci (gout associated) by the HOLMES method.
  • Figure 17 shows the detection of a SNP locus (gout associated) by the HOLMES method, in which the sample was a sample of 21 volunteers.
  • Figure 18 shows a scheme of primer design in one example of the invention that can be used for HOLMES detection of SNPs at any site.
  • Figure 19 uses a combination of LAMP and HOLMES to detect E. coli in the system.
  • A Electropherogram of the E. coli gyrB gene amplified by LAMP. A total of two sets of primers gyrB-1 and gyrB-2 were used for amplification. gyrB is a characteristic gene of Escherichia coli.
  • B The HOLMES detection system was used to detect the amplification product of LAMP. Negative control: the sample is sterile water, using gyrB-1 amplification primers to amplify or detect the results of the gyrB gene; gyrB-1: the sample is the E.
  • gyrB-2 the sample is the E. coli to be detected, and the second set of gyrB gene amplification primers is used to amplify or detect the result of the gyrB gene.
  • the rs5082 locus was detected using two crRNAs, crRNA-G and crRNA-T, respectively (SEQ ID NO: 5); rs1467558 was detected using two crRNAs, crRNA-C and crRNA-T, respectively. Listing 5).
  • Figure 21 uses RPA and HOLMES to jointly detect E. coli in the system.
  • A Amplification of the E. coli gyrB gene using RPA. A total of two sets of primers gyrB-1 and gyrB-2 were used for amplification. gyrB is a characteristic gene of Escherichia coli.
  • B The HOLMES detection system was used to detect the product of RPA amplification. Negative control: the sample is sterile water, using gyrB-1 amplification primers to amplify or detect the results of the gyrB gene; gyrB-1: the sample is the E.
  • gyrB-2 the sample is the E. coli to be tested, and the second set of gyrB amplification primers is used to amplify or detect the result of the gyrB gene.
  • Figure 22 Cas12b detection of single-stranded DNA as a target DNA by single-strand DNA cleavage activity. After completion of the bypass cleavage reaction, the reactants were separated by 12% urea denaturing gel electrophoresis and detected using a fluorescence imaging system. The numbers in parentheses represent the final concentration of the reactants in nM; the target DNA is a single-stranded DNA of 66 nt length in an amount of 50 nM; the single-stranded DNA probe is a single-stranded DNA with a FAM label at the 5' end, in an amount of 50 nM .
  • FIG. 23 Cas12b detection of single-stranded DNA and double-stranded DNA as a single-strand DNA cleavage activity of target DNA.
  • the reactants are detected using a fluorescent microplate reader.
  • the amount of Cas12b and guide RNA is 500nM;
  • the target DNA is single-stranded DNA or double-stranded DNA of 66nt in length, and the amount is 50nM;
  • the single-stranded DNA probe is a single-stranded DNA probe containing fluorescent reporter and quenching group (HEX -N12-BHQ1) in an amount of 500 nM.
  • both the single-stranded DNA template and the double-stranded DNA template can detect the viability of the bypass single-strand DNA cleavage after the addition of Cas12b and the guide RNA.
  • Figure 24 shows the bypass single-stranded DNA trans-cleavage activity of Cas12b against low concentrations of target DNA following binding to LAMP amplification.
  • the present inventors have developed a technical solution for target nucleic acid detection by extensively and intensively studying the cleavage properties of Cas enzymes such as Cas12a and Cas12b enzymes.
  • the experimental results show that the above technical solutions are successfully used to rapidly detect nucleic acids, for example, whether a certain concentration of microorganisms such as Escherichia coli and a rapid identification of SNP genotypes are identified.
  • the present invention has been completed on this basis.
  • guide RNA refers to an RNA that directs the Cas protein to specifically bind to a target DNA sequence.
  • crRNA refers to CRISPR RNA, which is a short RNA that directs Cas12a to bind to a target DNA sequence.
  • CRISPR refers to clustered regular interspaced short palindromic repeats, which are the immune systems of many prokaryotes.
  • Cas protein refers to a CRISPR-associated protein which is a related protein in the CRISPR system.
  • Cas12a refers to a crRNA-dependent endonuclease which is an enzyme of the V-A type (type V-A) in the CRISPR system classification.
  • Cas12b C2c1
  • CRISPR system classification The term “Cas12b”, “C2c1” is used interchangeably and refers to a crRNA-dependent endonuclease which is a V-B type (type V-B) enzyme in the CRISPR system classification.
  • LAMP is a loop-mediated isothermal amplification technique and is a thermostatic nucleic acid amplification technique suitable for gene diagnosis.
  • PAM refers to the protospacer-adjacent motif, which is required for Cas12a cleavage
  • the PAM of FnCas12a is the TTN sequence
  • the PAM of LbCas12a is the TTTN sequence
  • the PAM of AacCas12b is TTN.
  • the invention discloses a method for detecting a target nucleic acid molecule, which comprises a guide RNA, a Cas protein, a nucleic acid probe and a buffer in a reaction system containing a target nucleic acid molecule to be detected, and then performs fluorescence detection.
  • the Cas protein is Cas12a or Cas12b;
  • the Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; the Cas12a is preferably LbCas12a.
  • the Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b, AcCas12b.
  • the target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected is obtained after amplification.
  • the assay detects pathogenic microorganisms, genetic mutations or specific target DNA.
  • a use of a Cas protein in a method for detecting a target nucleic acid molecule is a use of a Cas protein in a method for detecting a target nucleic acid molecule.
  • the complex cleaves other single-stranded DNA molecules in the system.
  • the guide RNA refers to an RNA that directs the Cas protein to specifically target a DNA sequence.
  • the invention also provides a kit comprising a guide RNA, a Cas protein, a nucleic acid probe. Furthermore, the kit of the invention may further comprise a buffer.
  • the present invention provides a method for detecting a target nucleic acid molecule with high specificity and rapid detection. Once the target DNA (single or double stranded), the crRNA, and the Cas12a protein form a ternary complex, the complex cleaves other single stranded DNA molecules in the system.
  • the target DNA by designing crRNA (a DNA sequence to be detected); adding crRNA and Cas12a protein to the detection system; when the target DNA is present, Cas12a forms a ternary complex with the crRNA and the target DNA, and the complex exercises its Bypassing the activity of the cleavage and cleavage of the single-stranded DNA with fluorescent signal labeling (the luminescent group and the quenching group are respectively attached to the two ends, and the luminescent group can be luminescent after being cleaved), thereby emitting fluorescence. Therefore, by detecting the fluorescence, it is possible to know whether or not the target DNA molecule is contained in the system to be detected.
  • the method of the present invention allows rapid detection of whether a sample contains a specific DNA sequence. By combining with PCR technology, the sensitivity of the detection method can be greatly improved.
  • the nucleic acid probe in the present invention is preferably a fluorescent probe.
  • the invention provides the application of Cas12 enzyme based on Cas12a, Cas12b and the like in nucleic acid detection.
  • Cas12a as an example.
  • Cas12a has the activity of trans-cleavage (trans-cleavage), ie, when the target DNA, crRNA and Cas12a protein form a ternary complex, it will cleave other single-stranded DNA in the system (bypass single-stranded DNA) ).
  • trans-cleavage trans-cleavage
  • the bypass DNA was designed as a fluorescent probe consisting of a random sequence of 12 nt, and a fluorescent group HEX was labeled at the 5' end to label the quenching group BHQ1 (HEX-N12-BHQ1) at the 3' end.
  • a ternary complex of the target DNA, crRNA and Cas12a protein will be formed.
  • the probe is cleaved, and the HEX fluorescent group is emitted by the fluorescence detector to emit fluorescence. Light 535nM, emission light 556nM).
  • 10 different Cas12a were tested, and the target sequence was double-stranded DNA as shown in FIG. It can be seen that the complex composed of the target double-stranded DNA and each Cas12a protein can achieve trans-cleavage activity.
  • HOLMES response sensitivity Next, the sensitivity of FnCas12a and LbCas12a to target DNA was tested, that is, the concentration of the lowest target DNA that responded was examined. As shown in Fig. 9, when the test target was directly added, the target DNA above 0.1 nM was able to respond, and the response was significant at a concentration of 1 nM or more. If the PCR technique (HOLMES method) is combined, as shown in Fig. 8, the target fragment is first amplified by PCR and then subjected to Cas12a cleavage reaction. At this time, the response sensitivity can be as low as 10 aM, as shown in Fig. 9.
  • HOLMES method the PCR technique
  • SNP test Next, test whether the HOLMES method can detect the SNP genotype. T1 was used as the target sequence, and the PAM mutation or the 1-18 target sequence of the site was subjected to single point mutation, and the difference between the non-mutated sequence and the mutant sequence was compared between the crRNAs of different lengths.
  • the target complementary sequence is a 24 nt crRNA (crRNA-24 nt)
  • the single point mutation at 8-18 is not significantly different from the wild type, and the fluorescence value is after the PAM mutation and the 1-7 site mutation.
  • the fluorescence value of the 8-16 nt position at the mutation position was significantly lower than that of the target sequence of 24 nt.
  • the shortening was 16 nt or 17 nt, the fluorescence value of the target sequence after the mutation was observed.
  • the decrease is more obvious; when it is shortened to 15 nt, the fluorescence values of the target sequence and the mutation are weaker for the target sequence, but the intensity may still be higher for other target sequences and can be used for detection. Taken together, 15nt, 16nt and 17nt crRNAs are most suitable for the detection of SNPs.
  • Cas12a cleaves single-stranded DNA
  • Cas12a cleaves single-stranded DNA into a programmed cleavage mode independent of PAM sequence, called cis cleavage (cis cleavage); and once ternary complex Cas12a/crRNA/target DNA formation will show trans-cleavage (trans-cleavage) activity, ie, any single-stranded DNA that exhibits non-targets in the cleavage system.
  • HOLMES one HOur Low-cost Multipurpose Efficient Simple assay
  • the invention also provides a method of nucleic acid detection based on the bypass cleavage activity of a Cas12 enzyme, including Cas12a or Cas12b.
  • the detection of the invention can be performed on SNPs, in particular by PCR amplification followed by detection.
  • the synthesized crRNA based on the guide sequence designed according to the PAM site can be used for HOLMES detection.
  • the background signal is relatively low; for the same guide sequence, the signal difference between different SNP templates is relatively large.
  • a typical procedure involves designing a primer near the SNP site, carrying a PAM site on the primer, and the 3' end sequence at the PAM site needs to be paired with the template DNA.
  • the primer at the other end has no special requirements, and only needs to be able to be paired with the template DNA and can be subjected to PCR amplification.
  • the PAM site was successfully introduced after PCR amplification.
  • the SNP site is located at the position of the first 16 bases of the 5' end of the crRNA guide sequence, preferably 1-14, more preferably 1-12.
  • the bit is more preferably 1-11 or 1-10, most preferably 1-8 or 1-7.
  • the present invention has extremely high sensitivity and can detect DNA at a concentration of 10 aM.
  • Multi-purpose It can detect different nucleic acid samples, including DNA samples and RNA samples.
  • RNase inhibitor was purchased from TaKaRa, high-fidelity DNA polymerase KOD FX was purchased from ToYoBo; primer (oligonucleotide) was synthesized by Shanghai Biotech; T7 RNA polymerase was purchased from Thermo; RNA purification and concentration kit (RNA Clean&ConcentratorTM-5) was purchased from Zymo Research; SV Gel and PCR Clean-Up System were purchased from Promega; media (eg, Tryptone, Yeast Extract, etc.) were purchased from OXOID Corporation.
  • Target-T1-R Single-stranded DNA
  • a transcription template was prepared by annealing with T7-crRNA-F and a synthetic oligonucleotide T7-T1-24-R as shown in Table 5.
  • the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • the crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted to a concentration of 10 ⁇ M and stored in a -80 °C freezer.
  • Cas12a reaction In the 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), target single-stranded DNA (target-T1-R) (0.01 ⁇ M), nucleic acid probe (N25- 5' FAM) (0.01 ⁇ M), buffer was NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The blank control reaction was performed with the addition of other components, with no single-stranded DNA target sequence added. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection electrophoresis by urea-acrylamide gel electrophoresis (Urea-PAGE), followed by detection by a fluorescence luminescence imager.
  • different Cas12a have different detection effects on the target.
  • HkCas12a, etc. without the target single-stranded DNA will also cause the cutting of the probe.
  • LbCas12a, etc. only when the target single-stranded DNA is added, the cleavage of the probe occurs, which is a better candidate Cas12a protein.
  • Cas12a protein detection can detect single-stranded DNA targets (probes have HEX, BHQ1 double label)
  • Target-T1-R Single-stranded DNA
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C, diluted to a concentration of 10 [mu]M and stored in a -80 °C freezer.
  • Cas12a reaction In the 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), target single-stranded DNA (target-T1-R) (0.01 ⁇ M), fluorescent probe (HEX- N12-BHQ1, a 12 nt single-stranded DNA with a HEX tag at the 5' end and a BHQ1 tag at the 3' end (0.5 ⁇ M), a buffer of NEB buffer 3.1, 0.5 ⁇ L of RNase inhibitor. The other components of the control reaction were added, and only the single-stranded DNA target sequence was not added. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm).
  • a microplate reader excitation light 535 nm, emission light 556 nm.
  • different Cas12a have different detection effects on the target.
  • HkCas12a, etc. without the target single-stranded DNA will also cause the cutting of the probe.
  • FnCas12a et al. when the target single-stranded DNA is added, cleaves the probe, which is a better candidate Cas12a protein.
  • Target-T1 Double-stranded DNA (target-T1) was selected as the target sequence to test the response of different Cas12a proteins to their detection.
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C, diluted to a concentration of 10 [mu]M and stored in a -80 °C freezer.
  • Cas12a reaction In the 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), target double-stranded DNA (target-T1, from primer target-T1-F and primer target-T1- After R annealing (0.01 ⁇ M), fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer was NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 7, different Cas12a have different detection effects on the target. LbCas12a, etc., only when the target double-stranded DNA is added, the cleavage of the probe occurs, which is a better candidate Cas12a protein.
  • Target-T1 was selected as the target DNA, and then diluted to different concentrations to test the sensitivity of FnCas12a and LbCas12a. In order to increase the sensitivity, a PCR amplification step was added.
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C, diluted to a concentration of 10 [mu]M and stored in a -80 °C freezer.
  • PCR amplification was carried out by gradient dilution using a plasmid containing the target-T1 target (pUC18-T1) as a template.
  • the total volume of each reaction system was 20 ⁇ L, using 0.25 ⁇ M of M13F-47 and M13R-48 as primers (Table 4), and the PCR reaction using high-fidelity enzyme KOD FX (ToYoBo).
  • the PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After the PCR is completed, the PCR amplification product is directly used for the Cas12a reaction.
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, FnCas12a or LbCas12a (0.25 ⁇ M), PCR product 1 ⁇ L (or directly diluted into different concentrations of target DNA), fluorescent probe (HEX -N12-BHQ1) (0.5 ⁇ M), buffer was NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Fig. 9, when the test target was directly added, the target DNA above 0.1 nM was able to respond, and the response was significant at a concentration of 1 nM or more. If the PCR technique is combined, the target fragment is first amplified by PCR and the Cas12a cleavage reaction is performed. At this time, the response sensitivity can be as low as 10 aM.
  • Target-T1 was selected as the target, and it was subjected to single point mutation in the PAM region and 1-18, respectively, and the response values of several different lengths of crRNA to wild type and single point mutation were tested.
  • the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • the crRNA was synthesized using a T7 high yield transcription kit, and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C, diluted to a concentration of 10 [mu]M and stored in a -80 °C freezer.
  • PCR amplification A plasmid containing the target-T1 target (pUC18-T1) was used as a template. The total volume of each reaction system was 20 ⁇ L, and the PCR reaction was carried out using the high-fidelity enzyme KOD FX (ToYoBo) with 0.25 ⁇ M of the primer M13R-48 and each of the Target-T1-F mutant primers (Table 4). The PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After the PCR is completed, it is directly used for the Cas12a reaction.
  • KOD FX ToYoBo
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, FnCas12a or LbCas12a (0.25 ⁇ M), PCR product 1 ⁇ L, fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer The solution was NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm).
  • the target complementary sequence is a 24 nt crRNA (crRNA-24 nt)
  • the single point mutation at 8-18 is not significantly different from the wild type, and the fluorescence value is after the PAM mutation and the 1-7 site mutation. There is a significant drop.
  • the length of the trRNA was truncated, the length of the paired target sequence was 18 nt, and the fluorescence value of the 8-16 nt at the mutation position was significantly lower than that of 24 nt.
  • the fluorescence value of the target sequence after the mutation decreased more significantly, while at 15 nt, the fluorescence values of the target sequence and the mutation are already weak, but the intensity may still be high for other target sequences and can be used for detection. Taken together, 15nt, 16nt and 17nt crRNAs are most suitable for the detection of SNPs.
  • the E. coli gyrB gene was selected as the detection target to indirectly test the concentration of microorganisms such as Escherichia coli in the water.
  • Escherichia coli MG1655 was used as a positive control to determine the amount of microorganisms in the water (such as sewage and tap water) in the environment.
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • the crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified with NanoDrop 2000C, diluted to a concentration of 10 ⁇ M and stored in a -80 °C freezer.
  • PCR amplification The positive control sample was cultured to E. coli MG1655 until the OD 600 was about 0.5, and diluted as a template with a gradient of 10 times, and the sample was ambient water (including tap water and muddy water in the environment). The total volume of each reaction system was 20 ⁇ L, using 0.25 ⁇ M primers gyrB-F and gyrB-R (Table 4), and the PCR reaction was performed with the high-fidelity enzyme KOD FX (ToYoBo). The PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After PCR is complete, the PCR product is used directly for the Cas12a reaction.
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, LbCas12a (0.25 ⁇ M), PCR product 1 ⁇ L, fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was stopped at 37 ° C for 15 min and then at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 13, as the concentration of E. coli MG1655 decreased, its fluorescence response value gradually decreased. Among them, samples 2, 4, 5, and 6 were more obvious to detect microorganisms.
  • the SNP test selected 5 sites of human SNP, namely rs5082, rs1467558, rs2952768, rs4363657, rs601338, to test the feasibility of the HOLMES method.
  • a transcription template was prepared by annealing with T7-crRNA-F and a synthetic oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. Using RNA Clean & Concentrator TM -5 (Zymo Research) an RNA purified, and quantified using NanoDrop 2000C diluted to a concentration of 10 ⁇ M and stored at -80 °C freezer.
  • PCR amplification The total volume of the reaction system is 20 ⁇ L, using 0.25 ⁇ M primer (Table 4), 1 ng human genome (HEK293T) or directly scraping the oral epithelial mucosa as a template, and the PCR reaction using high-fidelity enzyme KOD FX (ToYoBo) ).
  • the PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After the PCR is completed, it is directly used for the Cas12a reaction.
  • the primers 1-rs5082-F-T, 2-rs1467558-F-T, 3-rs2952768-R-C directly introduce the corresponding mutation products of SNP
  • Cas12a reaction In the 20 ⁇ L reaction system, the corresponding crRNA (1 ⁇ M), LbCas12a (0.5 ⁇ M), PCR product 1 ⁇ L, and fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M) were added. The reaction was allowed to react at 37 ° C for 15 min and then stopped at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 14, only when the crRNA corresponds to the corresponding target sequence, there is a higher fluorescence response value, and if there is a mutation in one site, the response value is greatly reduced. The genotype of the corresponding SNP can be judged by the fluorescence value, and the results are confirmed by the sequencing results.
  • the TP53 gene was selected as a test gene in which a TP53 gene has a nonsense mutation in human T24 cells, resulting in inactivation of the gene.
  • the cells with normal gene (HEK293T), individual genetic tests, and mutant cell T24 were tested.
  • Preparation of crRNA First, by using T7-crRNA-F and the synthetic oligonucleotides T7-crRNA-34-TP53-T24-C-16nt and T7-crRNA-34-TP53-T24-G-16nt (Table 5) Annealing to prepare a transcription template. Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • PCR buffer Transgen Biotech
  • the crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. Using RNA Clean & Concentrator TM -5 (Zymo Research) an RNA purified, and quantified using NanoDrop 2000C diluted to a concentration of 10 ⁇ M and stored at -80 °C freezer.
  • PCR amplification The total volume of the reaction system is 20 ⁇ L, using 0.25 ⁇ M primer 34-TP53-T24-F, 34-TP53-T24-R (Table 4), 1 ng human genome (HEK293T, T24) or directly scraping
  • the oral epithelial mucosa was taken as a template, and the high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction.
  • the PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After the PCR is completed, it is directly used for the Cas12a reaction.
  • Cas12a reaction In the 20 ⁇ L reaction system, the corresponding crRNA (1 ⁇ M), LbCas12a (0.5 ⁇ M), PCR product 1 ⁇ L, and fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M) were added. The reaction was allowed to react at 37 ° C for 15 min and then stopped at 98 ° C for 2 min.
  • the SNP test used 5 sites of human SNP, which were related to the risk of gout, rs1014290, rs6449213, rs737267, rs1260326, rs642803, and tested the HOLMES method.
  • a transcription template was prepared by annealing with T7-crRNA-F and a synthetic oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was purified using RNA Clean & ConcentratorTM-5 (Zymo Research) and quantified with NanoDrop 2000C, diluted to a concentration of 10 ⁇ M and stored in a -80 °C freezer.
  • PCR amplification The total volume of the reaction system is 20 ⁇ L, using 0.25 ⁇ M primer (Table 4), 1 ng human genome (HEK293T) or directly scraping the oral epithelial mucosa as a template, and the PCR reaction using high-fidelity enzyme KOD FX (ToYoBo) ).
  • the PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s. After the PCR is completed, it is directly used for the Cas12a reaction.
  • the primers 1-rs5082-F-T, 2-rs1467558-F-T, 3-rs2952768-R-C directly introduce the corresponding mutation products of SNP
  • Cas12a reaction In the 20 ⁇ L reaction system, the corresponding crRNA (1 ⁇ M), LbCas12a (0.5 ⁇ M), PCR product 1 ⁇ L, and fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M) were added. The reaction was allowed to react at 37 ° C for 15 min and then stopped at 98 ° C for 2 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 16, only when the crRNA corresponds to the corresponding target sequence, there is a higher fluorescence response value, and if there is a mutation in one site, the response value is greatly reduced. The genotype of the corresponding SNP can be judged by the fluorescence value, and the results are confirmed by the sequencing results.
  • the premix was added to a 96-well plate, prepared into a kit, and then genomic DNA of 21 volunteers was added to test the rs1014290 site, which is associated with the risk of gout.
  • Kit Preparation First, a transcription template was prepared by annealing with T7-crRNA-F and a synthetic oligonucleotide (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute. The crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was purified using RNA Clean & ConcentratorTM-5 (Zymo Research) and quantified with NanoDrop 2000C and diluted to a concentration of 10 ⁇ M.
  • 96-well plate PCR premix In the 19 ⁇ L system, the reagents required for the PCR reaction were added, and the primers were 41-rs1014290-F and 41-rs1014290-R.
  • Fluorescence detection 96-well plate premix In a 19 ⁇ L system, add crRNA (1 ⁇ M), LbCas12a (0.5 ⁇ M), fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), and add to a 96-well plate.
  • PCR amplification Volunteer genomic DNA was added to a 96-well plate pre-mixed with PCR, followed by PCR reaction. The PCR reaction procedure was 95 ° C for 2 min, then 35 cycles of 98 ° C for 10 s, 60 ° C for 15 s, and 68 ° C for 10 s.
  • Cas12a reaction Take 1 ⁇ L of PCR reaction solution, add to the pre-mixed fluorescence detection 96-well plate, react at 37 ° C for 15 min, then terminate the reaction at 98 ° C for 2 min.
  • Fluorescence detection detection by a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 17, since the genotype A:A population has a higher risk of gout, except for volunteers 5, 7, and 9, other people are A:G or G:G genotypes, so more attention should be paid to the risk of gout. .
  • Example 11 Using LAMP to bind Cas protein to detect microorganisms such as Escherichia coli in environmental water
  • the E. coli gyrB gene was selected as the detection target to indirectly test whether microorganisms such as Escherichia coli were present in the water.
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ Taq DNA polymerase reaction buffer (Transgen Biotech) in a total volume of 50 ⁇ L, followed by an annealing procedure: initial denaturation at 95 ° C for 5 minutes, then from 95 °C was cooled to 20 ° C and lowered by 1 ° C per minute using a thermal cycler.
  • the crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using the RNA Purification and Concentration Kit and quantified using NanoDrop 2000C, finally diluted to a concentration of 10 ⁇ M and stored in a -80 ° C freezer for later use.
  • LAMP amplification sterile water and contaminated liquid containing E. coli were used as negative control and samples to be tested, respectively.
  • the total volume of each reaction system is 25 ⁇ L, and the kit for reacting LAMP-FIP with LAMP-BIP, 0.2 ⁇ M LAMP-F3 and LAMP-B3, 0.4 ⁇ M LAMP-LoopF and LAMP-LoopB, LAMP with primers for LAMP Kit (NEB).
  • the LAMP reaction procedure was at 65 ° C for 30 min. After the completion of LAMP, the fire was extinguished at 80 ° C for 10 min, and then directly used for the Cas12a reaction.
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), LAMP product 1 ⁇ L, fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was carried out at 37 ° C for 15 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). The result is shown in Figure 19.
  • LAMP amplification The human genome HEK293T was used as a sample. The total volume of each reaction system was 25 ⁇ L, using primers 1.6 ⁇ M of LAMP-FIP and LAMP-BIP, 0.2 ⁇ M of LAMP-F3 and LAMP-B3, 0.4 ⁇ M of LAMP-LoopF and LAMP-LoopB, and LAMP. LAMP Kit (NEB). The LAMP reaction procedure was at 65 ° C for 30 min. After the completion of LAMP, the fire was extinguished at 80 ° C for 10 min, and then directly used for the Cas12a reaction.
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), LAMP product 1 ⁇ L, fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was carried out at 37 ° C for 15 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). The result is shown in Figure 20.
  • Example 13 Amplification of Cas protein by RPA amplification to detect microorganisms such as Escherichia coli in environmental water
  • the E. coli gyrB gene was selected as the detection target to indirectly test whether microorganisms such as Escherichia coli were present in the water.
  • a transcription template was prepared by annealing with T7-crRNA-F and the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotide (4 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) in a total volume of 50 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • the crRNA was synthesized using the T7 high yield transcription kit and the reaction was carried out overnight (about 16 h) at 37 °C. RNA was then purified using an RNA purification and concentration kit and quantified using NanoDrop 2000C, finally diluted to a concentration of 10 ⁇ M and stored in a -80 ° C freezer for later use.
  • RPA amplification sterile water and Escherichia coli-contaminated liquid were used as negative control and samples to be tested. The total volume of each reaction system is 25 ⁇ L, and the reaction of RPA-gyrB-F (or RPA-gyrB-F2) with 0.5 ⁇ M primer and RPA-gyrB-R2, RPA is used. Basic kit (TwistDX). The RPA reaction procedure was 37 ° C for 30 min. After the completion of RPA, the fire was extinguished at 80 ° C for 10 min, and then directly used for the Cas12a reaction.
  • Cas12a reaction 20 ⁇ L reaction system, add the crRNA (0.5 ⁇ M) purified in step 1, Cas12a (0.25 ⁇ M), 1 ⁇ L of RPA product, fluorescent probe (HEX-N12-BHQ1) (0.5 ⁇ M), buffer NEB buffer 3.1, 0.5 ⁇ L RNase inhibitor. The reaction was carried out at 37 ° C for 15 min.
  • Fluorescence detection 20 ⁇ L of the inactivated reaction solution was added to a 96-well plate, and then detected by a microplate reader (excitation light 535 nm, emission light 556 nm). The result is shown in Figure 21.
  • the plasmid pUC18-guide RNA-T1 was constructed by using pUC18 as a plasmid backbone.
  • the plasmid inserted a T7 promoter and a template DNA sequence for transcription of the guide RNA on pUC18 (Note: The guide RNA transcribed from this template targets one The sequence referred to as T1 in this study).
  • the method was to use pUC18 plasmid as template, pUC18-1-F and pUC18-1-R as primers, first carry out a round of PCR, T4DNA Ligase was ligated into PCR product, transformed into DH10b, and sequenced to obtain correct clone, which is called pUC18- Guide RNA-T1-pre.
  • RNA-T1-pre The pUC18-guide RNA-T1-pre was used as a template, pUC18-2-F and pUC18-2-R were used as primers, and the second round of PCR was carried out. The PCR product was ligated and transformed in the same manner, and finally the correct plasmid pUC18- was obtained. Guide RNA-T1.
  • a guide RNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out overnight at 37 ° C (12-16 h).
  • target DNA is single-stranded
  • a 66 bp long oligonucleotide is directly synthesized as target DNA (target-T1-R), which contains the 20 bp target sequence (T1) recognized by the guide RNA.
  • target-T1-F target-T1-R
  • target-T1-R 20 bp target sequence
  • the paired oligonucleotide (1 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) to a total volume of 20 ⁇ L, and then subjected to an annealing procedure: initial denaturation at 95 ° C for 5 minutes, and then cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • Guide RNA annealing The guide RNA was diluted to an appropriate concentration (10 ⁇ M) and annealed in a PCR machine. Annealing procedure: denaturation at 75 ° C for 5 min, then cooling from 75 ° C to 20 ° C, 1 ° C per minute.
  • Cas12b reaction In a 20 ⁇ L reaction system, add the mixture of guide RNA and C2c1 incubated in step (2) (both concentrations are 250 ⁇ M or 500 ⁇ M), target DNA (final concentration 50 nM), FAM-labeled oligo Glycosidic acid (target-DNMT1-3-R-FAM-5') or fluorescence quenching probe (HEX-N12-BHQ1, final concentration 500 nM), and 2 ⁇ L of 10 ⁇ NEB Buffer 3.1 and 0.5 ⁇ L RNase inhibitor (40U/ ⁇ L). After mixing, the reaction was carried out at 48 ° C for 30 min. Thereafter, it was inactivated by heating at 98 ° C for 5 min in a PCR machine.
  • step (2) both concentrations are 250 ⁇ M or 500 ⁇ M
  • target DNA final concentration 50 nM
  • FAM-labeled oligo Glycosidic acid target-DNMT1-3-R-FAM-5'
  • fluorescence quenching probe HE
  • the inactivated 20 ⁇ L of the reaction solution was subjected to electrophoretic separation by urea denaturing gel electrophoresis, and then imaged using a fluorescence imaging system ImageQuant LAS 4000mini (GE Healthcare). The result is shown in Figure 22.
  • the inactivated 20 ⁇ L of the reaction solution was placed in a 96-well plate and detected by a microplate reader (excitation light 535 nm, emission light 556 nm). The result is shown in Figure 23.
  • the target DNA concentration required for Cas12b to perform trans-cleavage activity that is, the sensitivity of the Cas12b trans-cleavage reaction, was determined.
  • RNA-DNMT1-3-F and guide RNA-DNMT1-3-R as primers, and guide the guide RNA to 20 bases of target DNA of T1 by PCR.
  • guide RNA targeting DNMT1-3 another plasmid pUC18-guide RNA-DNMT1-3 was obtained.
  • a guide RNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out overnight at 37 ° C (12-16 h).
  • the first one is a reaction system directly added to Cas12b without amplification. Methods as below:
  • DNMT1-3 TTC PAM-R
  • DNMT1-3 target DNA
  • DNMT1-3 TTC PAM-F
  • DNMT1-3 TTC PAM-R
  • the identified 20 bp target sequence DNMT1-3.
  • the two oligonucleotides are annealed to give a short target DNA.
  • the paired oligonucleotide (2 ⁇ M) was annealed in 1 ⁇ PCR buffer (Transgen Biotech) to a total volume of 20 ⁇ L, and then an annealing procedure was performed: initial denaturation at 95 ° C for 5 minutes, followed by cooling from 95 ° C to At 20 ° C, use a thermal cycler to reduce 1 ° C per minute.
  • the single-stranded or double-stranded target DNA was serially diluted to 2 ⁇ M, 0.2 ⁇ M, 0.02 ⁇ M, 0.002 ⁇ M, and 0.0002 ⁇ M for use.
  • the second is to insert a fragment containing the target sequence (DNMT1-3) into a plasmid vector and amplify by LAMP reaction.
  • the target sequence (DNMT1-3) fragment was inserted into the pEasy-Blunt Zero Cloning Vector using Transgen's pEasy-Blunt Zero Cloning Kit, and the correct clone was obtained after sequencing.
  • the LAMP amplification reaction was carried out using the above plasmid as a template, and the template was added with 0,1 nM, 0.1 nM, and diluted 10 - fold to 10 -11 nM.
  • the total volume of each reaction system was 25 ⁇ L, using primers 1.6 ⁇ M of LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 ⁇ M of LAMP-DNM-F3 and LAMP-DNM-B3, and 0.4 ⁇ M of LAMP-DNM-LoopF.
  • LAMP-DNM-LoopB the kit used for the LAMP reaction is LAMP Kit (NEB).
  • the LAMP reaction procedure was at 65 ° C for 30 min. After the completion of LAMP, the fire was extinguished at 80 ° C for 10 min, and then directly used for the Cas12b reaction.
  • Guide RNA annealing The guide RNA was diluted to an appropriate concentration (5 ⁇ M) and annealed in a PCR machine. Annealing procedure: denaturation at 75 ° C for 5 min, then cooling from 75 ° C to 20 ° C, 1 ° C per minute.
  • the stem-loop structure of crRNA is important for stabilizing the structure of Cas12a, which is responsible for the necessity of cleavage of single-stranded DNA of Cas12a by the crRNA loop structure. It is further tested whether the single-stranded DNA cleavage site of Cas12a can pass through a shorter guide sequence, crRNA, such that the cleavage is outside the recognition site. At the length of the leader sequence of 16 nt, 18 nt and 20 nt, all of these crRNAs resulted in Cpf1 cleavage near the 22nd base, as shown in Figures 1B and 1D, meaning that the cleavage site is 4nt beyond the recognition site. , 2nt or 0nt.
  • the ternary complex also cleaves single-stranded DNA (ie, bypass single-stranded DNA) that does not have a complementary sequence to the crRNA in any other reaction system, as shown in Figures 3C and 3D.
  • This cutting phenomenon is a trans-cut, which is distinguished from a programmable cis-cut.
  • Trans-cleavage was also observed when the target single-stranded DNA was labeled at the 3'-end, but many cis-cleaving products were left, as shown in Figure 3B, which may be due to the formation of Cas12a/crRNA/target single-stranded DNA.
  • the complex, and the target single-stranded DNA is protected to protect the 3'-end of the tag from exposure to the nuclease active site of the ternary complex, and these cleavage processes can be shown in Figure 3A.
  • Trans-cleavage activity of the Cas12a complex In the structure of the Cas12b (ie C2c1) complex with additional single-stranded DNA, sequence-independent single-stranded DNA is also located on the surface of the catalytic pocket, as shown in Figure 6C, which is similar to Cas12a A bypass single-stranded DNA substrate. Combining single amino acid mutation experiments, the proposed target DNA, non-target DNA and bypass single-stranded DNA were all cut in a single RuvC pocket in Cas12a, as shown in Figures 6D, 6E and 6F.
  • the ternary Cas12a complex has bypass single-stranded DNA trans-cleavage activity, while the monomeric or binary complex does not bypass the single-stranded DNA trans-cleavage activity by comparing monomeric, binary, and ternary complexes.
  • the monomeric Cas12a structure is disordered
  • the binary complex Cas12a/crRNA is a triangular structure, as shown in Figure 6G
  • the ternary complex Cas12a/crRNA/target DNA is converted into a bilobal structure, thereby exposing the catalytic pocket to Trans-cutting of the bypass single-stranded DNA is achieved (as shown in Figure 6H).
  • HOLMES one HOur Low-cost Multipurpose Efficient Simple assay
  • the whole reaction system it can be divided into two large steps, one is the amplification of the template nucleic acid, and the other is the specific nucleic acid detection of the Cas12a protein.
  • PCR is used for amplification of nucleic acids, but in practice, any amplification method can be combined with nucleic acid detection of the second step, such as isothermal amplification method RPA or the like.
  • the initial nucleic acid is not limited to double-stranded DNA, and may be single-stranded DNA; even RNA can be detected by reverse transcription, and thus the method is applicable to various types of nucleic acid molecules.
  • the nucleic acid detection phase three of the components are the key to the experiment, namely Cas12a, crRNA and nucleic acid probes.
  • the crRNA as a guiding effect is more stable in the system after being modified by artificial modification or the like.
  • the present invention selects HEX and BHQ1 labeled short single stranded DNA, and any other detectable labeling method is theoretically applicable as long as the nucleic acid probe is cleaved to produce a detectable difference.
  • the nucleic acid probe can be designed to fluoresce after binding to the compound to detect if the probe is cleaved.
  • LAMP amplified DNA binds to the primer used for Cas12a detection:

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Abstract

本发明提供了一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒，靶标核酸分子的检测方法包括向含有待检靶标核酸分子的反应体系中加入向导RNA、Cas12a、核酸探针，反应完成后对其进行检测。

Description

一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒 技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种用于靶标核酸分子检测的方法。
背景技术
特异性检测核酸分子(Nucleic acid detection)方法具有重要的应用价值，例如病原体的检测，遗传病检测等。在病原体检测方面，由于每种病原体微生物都有其独一无二的特征核酸分子序列，因此可以开发出针对特定物种的核酸分子检测，也称为核酸诊断(NADs,nucleic acid diagnostics)，在食品安全、环境微生物污染检测，人体病原菌感染等领域具有重要意义。另一个方面是对人或其他物种的单核苷酸多态性(SNPs,single nucleotide polymorphisms)的检测。在基因组水平上去理解遗传变异和生物学功能之间的关系为现代分子生物学提供了新视角，而其中SNPs对生物学的功能、进化和疾病等密切相关，因此SNPs的检测与分析技术的发展尤为重要。
目前已建立了不少NADs的方法，主要是针对特异性DNA分子的检测，也有部分方法针对RNA分子。通常来说，DNA分子非常稳定，因此检测样品可来源于一系列复杂的生物样本；而RNA则非常容易降解，因此处理时需要非常小心。在上世纪70年代，建立了限制性内切酶酶切检测的方法，后来又发展了Southern、Northern和斑点杂交等方法进行特异性检测核酸分子检测。1985年，当PCR方法成为常规实验方法后，导致了分子生物学指数级的进步。目前建立的特异性核酸分子检测通常需要分为两步，第一步是目的核酸的扩增，第二步是目的核酸检测。PCR技术是最先建立也是目前最常用的扩增方法，目前在PCR方法基础上，引入了荧光标记的探针，可以实时检测靶标的扩增情况，称为Realtime PCR。Realtime PCR不仅是快速、高灵敏的检测方法，同时这种方法也可以进行定量分析。除了PCR的扩增方法，还建立了许多替代方法，比如ligase chain reaction,branched DNA amplification,NASBA,SDA,transcription-mediated amplification,Loop-mediated isothermal amplification(LAMP),rolling circle amplification(RCA)and Recombinase Polymerase Amplification(RPA)等。许多这些替代方法的优势在于等温性，也就是说只需要一个温度即可以完成反应，而不需要像PCR那样的热循环仪器。核酸检测的方法除了Realtime PCR可以直接完成扩增和检测之外，FISH杂交技术(Fluorescence in situ hybridization)是 最常用的检测方法——通过标记分子探针，原位与互补的靶标序列杂交。除此之外，目前还开发了下一代测序技术(next-generation sequencing technologies)和Oxford Nanopore测序技术等检测方法，但这些方法通常需要昂贵的实验设备。
对SNPs的检测首先同样需要进行PCR等方法的扩增，从而获得足量的含SNP位点区域片段进行进一步的检测。比较常用的方法有：引物延伸(primer extension)，杂交(hybridization)，连接(ligation)和酶切(enzymatic cleavage)。当完成上述方法之后，需要利用特定的方法进行检测，比如质谱检测、荧光检测、化学发光检测等。
核酸检测虽然如上文所述已开发出了不少检测方法，但是在某些情况下，如何更加快速、简便、廉价的检测仍是重要的发展方向，比如在野外的病原菌快速检测、药物敏感SNP快速检测等。在2016年，Collins等基于CRISPR-Cas9特异性识别并切割靶标序列的特点，开发了快速廉价检测寨卡病毒(Zika)的方法。2017年，张锋等利用CRISPR-Cas13a具有“旁路活性”(collateral effect)的特点建立了快速核酸探测的方法。“旁路活性”即Cas13a结合特异性靶标RNA后随机切割其他非靶标RNA(这里将RNA分子设计成RNA荧光报告系统)；通过与等温扩增技术RPA相结合，以实现快速靶标RNA的检测；张锋团队把这种检测方法称为SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing)。SHERLOCK方法涉及到RNA模板的结合，因此如果需要针对DNA进行检测，则需要先将DNA转录为RNA模板以用于检测；鉴于RNA的不稳定性，该方法无疑会增加操作的困难程度。
2015年，张锋等人发现了新的CRISPR相关蛋白内切酶Cas12a(之前称为Cpf1)，它与常用的Cas9蛋白一样是RNA引导的特异性DNA核酸内切酶；但与相比Cas9，Cas12a又有它自身特点，比如仅需要crRNA即可引导特异性切割双链DNA，并产生粘性末端等。
发明内容
本发明的目的在于提供一种靶标核酸分子的检测方法。
本发明的另一目的是提供一种Cas蛋白在靶标核酸分子的检测方法中的用途。
在本发明第一方面，提供了一种试剂盒，包括向导RNA、Cas蛋白、核酸探针、缓冲液。
一种靶标核酸分子的检测方法，将含有待检靶标核酸分子的反应体系中，加入向导RNA、Cas蛋白、核酸探针、缓冲液，然后对其进行检测(尤其是通过检测荧光强度的方法进行检测)。
优选地，所述Cas蛋白为Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白。
优选地，所述Cas蛋白为Cas12a。
所述的Cas12a优选为FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a中一种。
优选地，所述的Cas12a为LbCas12a。
优选地，向导RNA是指引导Cas蛋白特异性结合靶标DNA的RNA。
在另一优选例中，所述核酸探针为单链DNA；所述的单链DNA优选为荧光标记的单链DNA；所述的单链DNA优选为在5’端标记荧光基团HEX并在3’端标记淬灭基团BHQ1后的荧光探针。
在另一优选例中，所述核酸探针的检测方法优选为荧光检测法；所述的荧光检测法优选为使用酶标仪或者荧光分光光度计进行检测的方法。
优选地，待检靶标核酸分子的反应体系中的待检靶标核酸分子经过扩增后得到。
优选地，本发明的检测方法可检测病原微生物、基因突变或特异靶标DNA
在另一优选例中，所述的Cas蛋白包括Cas12b(C2c1)。
在本发明的第二方面，提供了一种Cas蛋白在靶标核酸分子的检测方法中的用途，或用于制备一制剂，所述制剂用于检测靶标核酸分子。
在另一优选例中，靶标DNA、向导RNA和Cas蛋白形成三元复合体时，该复合物会切割体系中其它的单链DNA分子。
优选地，向导RNA是指引导Cas蛋白特异性结合靶标DNA的RNA。
在本发明的第三方面，提供了一种试剂盒，包括向导RNA、Cas蛋白、核酸探针。
在另一优选例中，所述试剂盒还包括缓冲液。
在本发明的第四方面，提供了一种用于检测靶标核酸分子的检测体系，该体系包含：
(a)Cas蛋白，所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白；
(b)向导RNA，所述向导RNA引导Cas蛋白特异性结合于靶标核酸分子；和
(c)核酸探针，所述核酸探针为单链DNA；
其中，所述的靶标核酸分子为靶标DNA。
在另一优选例中，所述的检测体系还含有(d)缓冲液。
在另一优选例中，所述的检测体系还含有待检测的靶标核酸分子。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1-100拷贝/微升或10 15拷贝/微升，较佳地1-10拷贝/微升，更佳地1-5拷贝/微升。
在另一优选例中，所述的检测体系中，所述核酸探针与所述靶标核酸分子的摩尔比为10 3：1至10 14：1，较佳地10 4：1至10 7：1。
在另一优选例中，所述的靶标核酸分子的检测位点位于所述向导RNA的PAM序列下游第1-12位。
在另一优选例中，所述的向导RNA的长度为15-30nt，较佳地15-18nt。
在另一优选例中，所述的靶标DNA包括cDNA。
在另一优选例中，所述的靶标DNA选自下组：单链DNA、双链DNA、或其组合。
在另一优选例中，所述的核酸探针带有荧光基团和淬灭基团。
在另一优选例中，所述的荧光基团和淬灭基团各自独立地位于所述核酸探针的5’端、3’端和中部。
在另一优选例中，所述的核酸探针的长度为3-300nt，较佳地5-100nt，更佳地6-50nt，最佳地8-20nt。
在另一优选例中，所述靶标核酸分子包括来源于选自下组的靶标核酸分子：植物、动物、昆虫、微生物、病毒、或其组合。
在另一优选例中，所述的靶标DNA是人工合成或天然存在的DNA。
在另一优选例中，所述的靶标DNA包括野生型或突变型的DNA。
在另一优选例中，所述的靶标DNA包括由RNA逆转录或扩增而获得的DNA，如cDNA等。
在另一优选例中，所述的Cas12a选自下组：FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a或其组合；更佳地，所述的Cas12a为LbCas12a。
在另一优选例中，所述的具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白选自下组：Cas12b(即C2c1)。
在另一优选例中，所述的Cas12b蛋白选自下组：AacCas12b(Alicyclobacillus acidoterrestris)、Aac2Cas12b(Alicyclobacillus acidiphilus)、AkCas12b(Alicyclobacillus kakegawensis)、AmCas12b(Alicyclobacillus  macrosporangiidus)、AhCas12b(Alicyclobacillus herbarius)、AcCas12b(Alicyclobacillus contaminans)。
在另一优选例中，所述的核酸探针包括带有可检测标记的单链DNA。
在另一优选例中，所述的单链DNA为荧光和生物素标记的单链DNA。
在另一优选例中，所述的单链DNA为荧光标记的单链DNA。
在另一优选例中，所述的单链DNA为在5’端标记荧光基团HEX并在3’端标记淬灭基团BHQ1后的荧光探针。
在本发明的第五方面，提供了一种用于检测靶标核酸分子的试剂盒，所述试剂盒包括：
i)第一容器以及位于第一容器内的Cas蛋白，所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白；
ii)任选的第二容器以及位于第二容器内的向导RNA，所述向导RNA引导所述Cas蛋白特异性结合于靶标核酸分子；
iii)第三容器以及位于第三容器内的核酸探针；
iv)任选的第四容器以及位于第四容器内的缓冲液；
其中，所述的靶标核酸分子为靶标DNA。
在另一优选例中，所述的第一容器、第二容器、第三容器和第四容器中的任何二个、三个、或四个(或全部)可以是相同或不同容器。
在另一优选例中，所述的核酸探针带有荧光基团和淬灭基团。
在本发明的第六方面，提供了一种检测样本中是否存在靶标核酸分子的方法，其特征在于，包括以下步骤：
(a)提供本发明第四方面所述的用于检测靶标核酸分子的检测体系，并且所述的检测体系还具有待检测的样本；和
(b)检测所述检测体系中的核酸探针是否被Cas蛋白进行切割，所述的切割为旁路单链DNA的反式切割；
其中，所述核酸探针被Cas蛋白切割，则表示所述样本中存在靶标核酸分子；而所述核酸探针不被Cas蛋白切割，则表示所述样本中不存在靶标核酸分子。
在另一优选例中，所述的待检测的样本包括未经扩增的样本以及经过扩增(或核酸扩增)的样本。
在另一优选例中，所述的待检测的样本是经过扩增而获得的样本。
在另一优选例中，所述核酸扩增的方法选自下组：PCR扩增、LAMP扩增、RPA扩 增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA,SPIA,NEAR,TMA和SMAP2。
在另一优选例中，所述的PCR包括高温PCR、常温PCR、低温PCR。
在另一优选例中，所述方法用于检测靶位点处的核酸是否在SNP、点突变、缺失、和/或插入。
在另一优选例中，当所述的靶位点的上下游(-20nt至+20nt范围内，较佳地-15nt至+15nt范围内，更佳地-10nt至+10nt范围内)缺乏PAM序列时，采用引入PAM的引物进行核酸扩增。
在另一优选例中，所述的引入PAM的引物具有从5’-3’的式I结构：
P1-P2-P3  (I)
式中，
P1为位于5’端的与靶标核酸分子的序列互补或非互补的5’区段序列；
P2为PAM序列；
P3为位于3’端的与靶标核酸分子的序列互补3’区段序列。
在另一优选例中，所述的PAM引物特异性结合于靶标核酸分子的上游或下游。
在另一优选例中，P1的长度为0-20nt。
在另一优选例中，P3的长度为5-20nt。
在另一优选例中，所述的PAM引物的长度为18-50nt，较佳地20-35nt。
在另一优选例中，所述的互补包括完全互补和部分互补。
在另一优选例中，所述的核酸扩增中使用至少一条引物含有PAM序列。
在另一优选例中，当所述的靶位点的上下游(-20nt至+20nt范围内，较佳地-15nt至+15nt范围内，更佳地-10nt至+10nt范围内)含有PAM序列时，则可采用含有或不含有PAM序列的引物，并且扩增出的扩增产物含有所述PAM序列。
在另一优选例中，在步骤(b)中的检测包括荧光检测法。
在另一优选例中，所述荧光检测法采用酶标仪或者荧光分光光度计进行检测。
在本发明的第七方面，提供了一种Cas蛋白的用途，它被用于制备基于旁路单链DNA切割检测靶标核酸分子的检测试剂或试剂盒，其中所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白。
在另一优选例中，所述的Cas12a选自下组：FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a或其组合；更佳地，所述的Cas12a为LbCas12a。
在另一优选例中，所述的具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白选自下组：Cas12b(or C2c1)。
在另一优选例中，所述的Cas12b蛋白选自下组：AacCas12b。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Cas12a切割靶标单链DNA的cis切割(顺式切割)特性。
图2显示了Cas12a对靶标单链DNA切割时不依赖于切割双链时所需的PAM序列。
图3显示了Cas12a切割单链DNA的trans切割(反式切割)特性。
图4显示了测试的10种不同来源的Cas12a，这些Cas12a都具有单链DNA的顺式切割和反式切割活性。
图5通过Cas12a的单位点突变实验，鉴定了Cas12a中与单链DNA的顺式切割和反式切割活性可能相关的位点。
图6显示了Cas12a与Cas12b(即C2c1)单体及其与向导RNA、靶标DNA的复合体的结构。
图7显示了不同Cas12a利用特异性双链DNA底物，以单链DNA(HEX-N12-BHQ1)作为荧光检测探针，得到的荧光数值。阴性对照组不加特异性底物。
图8显示了基于靶标DNA扩增和Cas12a针对旁路单链DNA反式切割活性的HOLMES方法用于检测靶标DNA的流程示意图。
图9显示了直接利用FnCas12a或LbCas12a，或结合HOLMES方法的靶标DNA的灵敏度测试。
图10显示了不同长度向导序列的crRNA结合FnCas12a或LbCas12a对不同单位点突变的靶标序列的HOLMES法荧光检测值。
图11利用FAM标记的荧光探针，选用10种Cas12a蛋白测试加入靶标单链DNA之后，FAM标记的单链DNA探针是否被反式切割。
图12利用HEX-N12-BHQ1为探针，选用10种Cas12a蛋白测试加入靶标单链DNA之后，测试荧光值。
图13(A)利用HEX、BHQ1进行两端标记的单链DNA荧光探针显示了以gyrB基因片段为靶标序列，以不同浓度的纯培养大肠杆菌MG1655为正对照模板的HOLMES检测值。显示了随着大肠杆菌MG1655浓度的减少，其荧光响应值逐渐减少。(B) 不同地点环境中水样的检测值。
图14显示了HOLMES方法检测SNP的流程示意图，以及5个SNP位点的荧光检测值。
图15显示了HOLMES方法检测TP53基因(癌症相关基因)中关键位点的荧光检测值。
图16显示了HOLMES方法检测5个SNP位点(痛风相关)的检测值。
图17显示了HOLMES方法检测1个SNP位点(痛风相关)的检测值，其中样本为21名志愿者样本。
图18显示了本发明一个实例中的引物设计的方案，可用于针对任意位点的SNP进行HOLMES检测。
图19利用LAMP和HOLMES联合检测体系中大肠杆菌。(A)利用LAMP扩增大肠杆菌gyrB基因的电泳图。总共使用了两组引物gyrB-1和gyrB-2来进行扩增。gyrB为大肠杆菌的特征基因。(B)利用HOLMES检测体系来检测LAMP的扩增产物。阴性对照：样本为无菌水，使用gyrB-1扩增引物来扩增或检测gyrB基因的结果；gyrB-1：样本为待检测大肠杆菌，使用第一组gyrB基因扩增引物来扩增或检测gyrB基因的结果；gyrB-2：样本为待检测大肠杆菌，使用第二组gyrB基因扩增引物来扩增或检测gyrB基因的结果。
图20利用LAMP和HOLMES联合检测人HEK293T细胞的基因型。(A)利用LAMP扩增人HEK293T细胞相应SNP检测模板的电泳图。阴性对照：样本为无菌水，使用rs5082扩增引物的扩增结果；rs5082：样本为人HEK293T细胞的总基因组，使用rs5082扩增引物的扩增结果；rs1467558：样本为人HEK293T细胞的总基因组，使用rs1467558扩增引物的扩增结果。(B)利用HOLMES检测体系来检测LAMP的扩增产物。rs5082位点的检测分别使用了crRNA-G和crRNA-T两种crRNA来进行检测(序列表5)；rs1467558位点的检测分别使用了crRNA-C和crRNA-T两种crRNA来进行检测(序列表5)。
图21利用RPA和HOLMES联合检测体系中大肠杆菌。(A)利用RPA扩增大肠杆菌gyrB基因。总共使用了两组引物gyrB-1和gyrB-2来进行扩增。gyrB为大肠杆菌的特征基因。(B)利用HOLMES检测体系来检测RPA扩增的产物。阴性对照：样本为无菌水，使用gyrB-1扩增引物来扩增或检测gyrB基因的结果；gyrB-1：样本为待检测大肠杆菌，使用第一组gyrB扩增引物来扩增或检测gyrB基因的结果；gyrB-2：样本为待检测大肠杆菌，使用第二组gyrB扩增引物来扩增或检测gyrB基因的结果。
图22 Cas12b以单链DNA作靶标DNA的旁路单链DNA切割活力检测。在旁路切割反应完成后，反应物经过12％尿素变性凝胶电泳分离，并使用荧光成像系统进行检测。括号内的数字代表反应物的终浓度，单位为nM；靶标DNA为66nt长度 的单链DNA，用量为50nM；单链DNA探针为5’端带有FAM标记的单链DNA，用量为50nM。从图中可以看出，在含有Cas12b、向导RNA、靶标DNA之后，FAM标记的单链DNA被切成碎片，即Cas12b具有旁路单链DNA切割活力。
图23 Cas12b以单链DNA和双链DNA作靶标DNA的旁路单链DNA切割活力检测。在旁路切割反应完成后，反应物利用荧光酶标仪进行检测。Cas12b和向导RNA的用量均为500nM；target DNA为长66nt的单链DNA或双链DNA，用量为50nM；单链DNA探针为含荧光报告和淬灭基团的单链DNA探针(HEX-N12-BHQ1)，用量为500nM。从图中可以看出，无论是单链DNA模板还是双链DNA模板，在添加了Cas12b和向导RNA之后，均可以检测出旁路单链DNA切割的活力。
图24显示了Cas12b在结合了LAMP扩增之后针对低浓度的靶标DNA产生的旁路单链DNA反式切割活力。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明人通过广泛而深入的研究，通过对Cas酶(如Cas12a和Cas12b酶)的切割特性的研究，开发了一种靶标核酸检测的技术方案。实验结果表明，采用上述技术方案成功地快速检测核酸，例如鉴定了水体中是否含有一定浓度的大肠杆菌等微生物，以及SNP基因型的快速鉴定。在此基础上完成了本发明。
术语
术语“向导RNA”是指引导Cas蛋白特异性结合靶标DNA序列的RNA。
术语“crRNA”是指CRISPR RNA，是短的引导Cas12a到结合到靶标DNA序列的RNA。
术语“CRISPR”是指成簇的、规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats)，该序列是许多原核生物的免疫系统。
术语“Cas蛋白”是指CRISPR-associated蛋白，它是CRISPR系统中的相关蛋白。
术语“Cas12a”(旧称“Cpf1”)是指crRNA依赖的内切酶，它是CRISPR系统分类中V-A型(type V-A)的酶。
术语“Cas12b”，“C2c1”可互换使用，是指crRNA依赖的内切酶，它是CRISPR系统分类中V-B型(type V-B)的酶。
术语“LAMP”是环介导等温扩增技术(Loop-mediated isothermal amplification),是一种适用于基因诊断的恒温核酸扩增技术。
术语“PAM”是指前间区序列邻近基序(protospacer-adjacent motif)，是Cas12a切割所必须，FnCas12a的PAM为TTN序列，LbCas12a的PAM为TTTN序列，AacCas12b的PAM为TTN。
本发明公开了一种靶标核酸分子的检测方法，将含有待检靶标核酸分子的反应体系中，向导RNA、Cas蛋白、核酸探针、缓冲液，然后对其进行荧光检测。
所述Cas蛋白为Cas12a或者Cas12b；
所述的Cas12a优选为FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a中一种；所述的Cas12a优选为LbCas12a。
所述的Cas12b优选为AacCas12b、Aac2Cas12b、AkCas12b、AmCas12b、AhCas12b、AcCas12b。
向导RNA是指引导Cas蛋白特异性靶向DNA序列的RNA。
待检靶标核酸分子的反应体系中的待检靶标核酸分子经过扩增后得到。
该检测方法可检测病原微生物、基因突变或特异靶标DNA。
一种Cas蛋白在靶标核酸分子的检测方法中的用途。
靶标DNA、向导RNA和Cas蛋白形成三元复合体时，该复合物会切割体系中其他的单链DNA分子。
向导RNA是指引导Cas蛋白特异性靶向DNA序列的RNA。
本发明还提供了一种试剂盒，包括向导RNA、Cas蛋白、核酸探针。此外，本发明试剂盒还可包括缓冲液。
本发明提供了一种高特异性地快速检测靶标核酸分子的检测方法。一旦靶标DNA(单链或者双链)、crRNA和Cas12a蛋白形成三元复合体时，该复合物会切割 体系中其他的单链DNA分子。通过设计crRNA靶向靶标DNA(需要检测的一段DNA序列)；向检测体系中加入crRNA和Cas12a蛋白；当靶标DNA存在时，Cas12a与crRNA以及靶标DNA形成三元复合体，同时该复合物行使其旁路切割的活性并切割带荧光信号标记的单链DNA(两头分别连有发光基团和淬灭基团，被切断后发光基团可以发光)，从而发出荧光。因此，通过检测荧光即可得知待检测体系中是否含有靶标DNA分子。使用本发明的方法可快速检测样品中是否含有特异DNA序列。通过与PCR技术的结合，该检测方法的灵敏度可以得到大幅度提高。本发明中的核酸探针优选为荧光探针。
HOLMES条件测试：
本发明提供了基于Cas12a、Cas12b等Cas12酶在核酸检测中的应用。以下以Cas12a为例进行描述。
Cas12a的选择：根据研究，Cas12a具有trans切割(反式切割)的活力，即一旦靶标DNA、crRNA和Cas12a蛋白形成三元复合体时，会切割体系中其他的单链DNA(旁路单链DNA)。根据这一原理设计了特异性DNA检测方法。首先，将旁路DNA设计成荧光探针，其组成是12nt的随机序列，并在5’端标记荧光基团HEX在3’端标记淬灭基团BHQ1(HEX-N12-BHQ1)。当体系中含有标靶DNA片段时，将形成靶标DNA、crRNA和Cas12a蛋白的三元复合体，此时该探针即被切割，同时通过荧光检测仪的检测HEX荧光基团会发出荧光(激发光535nM，发射光556nM)。接着，测试了10种不同的Cas12a，靶标序列为双链DNA如图7所示。可以看出，靶标双链DNA与各个Cas12a蛋白组成的复合体都能够实现trans切割活力。
HOLMES响应灵敏度：接着，测试了FnCas12a与LbCas12a对靶标DNA的响应灵敏度，也就是说考察能响应的最低靶标DNA的浓度。如图9所示，当直接加入测试靶标时，0.1nM浓度以上的靶标DNA都能够响应，而且浓度1nM以上时响应显著。如果结合PCR技术(HOLMES方法)，如图8所示，即先通过PCR扩增目的片段再进行Cas12a切割反应，此时，响应灵敏度可以低至10aM，如图9所示。
SNP测试：接着，测试HOLMES方法是否可以检测SNP基因型。将T1作为靶标序列，将该位点的PAM突变或1-18位靶标序列分别进行单位点突变，比较不同长度的crRNA对非突变序列和突变序列之间测差异。
如图10所示，当靶标互补序列为24nt的crRNA(crRNA-24nt)时，8-18位的单位点突变与野生型差异不大，而PAM突变和1-7位点突变后，荧光值有明显的 下降。当截短crRNA长度，配对靶标序列长度为18nt时，突变位置8-16nt的荧光值相比靶标序列长度为24nt时有明显下降；当继续缩短为16nt或17nt时，突变后的靶标序列荧光值的下降幅度更加明显；而再缩短为15nt时，靶标序列和突变的荧光值针对该靶标序列比较弱，但是针对其他的靶标序列时其强度可能仍会较高，可以用于检测。综合来看，15nt、16nt和17nt的crRNA对SNP的检测最为合适。
本发明对Cas12a切割单链DNA，Cas12a对单链DNA的切割是不依赖于PAM序列的程序化切割方式，称之为cis切割(顺式切割)；而一旦三元复合体Cas12a/crRNA/靶标DNA形成,就会显示出trans切割(反式切割)的活性，即会表现出切割体系中非靶标的任意单链DNA。
利用Cas12a的特性，开发了特异性检测核酸分子的方法，称之为HOLMES(one HOur Low-cost Multipurpose Efficient Simple assay)。正如该技术的名称一样，其特点是快速(1小时)、低价、多通路、高效、简便测试法。该方法可用于快速病原菌检测，SNP检测等领域。
基于旁路切割活性进行核酸检测
本发明还提供了基于Cas12酶(包括Cas12a或Cas12b)的旁路切割活性进行核酸检测的方法。
优选地，本发明的检测可针对SNP进行，尤其是先进行PCR扩增，然后进行检测。
参见图18，其中给出了引物的设计方案。
情形1.当SNP位点附近有PAM位点，且依据该PAM位点设计的guide序列基础上合成的crRNA能够用于HOLMES检测。当用HOLMES方法进行检测时，其表现为背景信号比较低；针对同一条guide序列，不同SNP模板之间的信号差异比较大。
情形2.当SNP位点附近没有PAM位点，或者没有合适的PAM位点时，可以依据以上实验方案进行PAM位点的引入。
一种典型的步骤包括：在SNP位点附近设计引物，在引物上携带PAM位点，在PAM位点的3’端序列需要与模板DNA配对。另一端的引物没有特殊要求，只需要能够与模板DNA进行配对并能够进行PCR扩增即可。如图18所示，在进行PCR扩增之后，PAM位点便能够被成功引入。
参见图10，在本发明中，在设计引入PAM位点时，通常SNP位点位于crRNA guide序列5’端的前16个碱基的位置，较佳地1-14位，更佳地1-12位，更优选为1-11位或1-10位，最佳地1-8位或1-7位。
本发明的主要优点在于：
(1)快速：在测试条件准备好的情况下，从拿到样品，到拿到检测结果只需约1小时。
(2)低成本：实验中没有特殊的材料或酶，而且涉及到的材料、试剂等较少，可以进行微量化的测试分析。
(3)高效：本发明具有极高的灵敏度，可以检测到10aM浓度的DNA。
(4)多用途：可检测不同的核酸样本，包括DNA样本和RNA样本。
(5)简单：没有特殊复杂的步骤，如果制成试剂盒以及设定好程序，只需简单的加入样品等操作。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
材料
1.RNA酶抑制剂购自TaKaRa公司，高保真DNA聚合酶KOD FX购自ToYoBo公司；引物(寡核苷酸)由上海生工合成；T7RNA聚合酶购自Thermo公司；RNA纯化与浓缩试剂盒(RNA Clean&ConcentratorTM-5)购自Zymo Research；
Figure PCTCN2018082769-appb-000001
SV Gel and PCR Clean-Up System购自Promega公司；培养基(如，Tryptone，Yeast Extract等)均购自OXOID公司。
2.培养基配方：液体LB(1％Tryptone，0.5％Yeast extract,1％NaCl)，配置固体LB时，只需要在液体LB中添加2％的琼脂即可。
实施例1 Cas12a蛋白检测可探测单链DNA靶标(探针FAM标记)
选用单链DNA(target-T1-R)作为靶标序列，测试不同Cas12a蛋白对其检测的响应值。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-T1-24-R，如表5所示，进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C(Thermo Fisher Scientific)定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，靶标单链DNA(target-T1-R)(0.01μM)，核酸探针(N25-5′FAM)(0.01μM)，缓冲液为NEB buffer 3.1，0.5μLRNA酶抑制剂。空白对照反应为其他成分都加，仅不加单链DNA靶标序列。在37℃反应15min，然后98℃2min终止反应。
3、荧光检测：通过尿素-丙烯酰胺凝胶电泳(Urea-PAGE)电泳，然后用荧光发光成像仪检测。如图11所示，不同Cas12a对靶标检测效果不同。如HkCas12a等，不加靶标单链DNA也会造成探针的切割。而LbCas12a等，只在加入靶标单链DNA时，发生探针的切割，是较好的候选Cas12a蛋白。
实施例2 Cas12a蛋白检测可探测单链DNA靶标(探针有HEX、BHQ1双标记)
选用单链DNA(target-T1-R)作为靶标序列，测试不同Cas12a蛋白对其检测的响应值。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-T1-24-R(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，靶标单链DNA(target-T1-R)(0.01μM)，荧光探针 (HEX-N12-BHQ1，即12nt的单链DNA，其5’端为HEX标记，3’端为BHQ1标记)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。对照反应其他成分都加，仅不加单链DNA靶标序列。在37℃反应15min，然后98℃2min终止反应。
3、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图12所示，不同Cas12a对靶标检测效果不同。如HkCas12a等，不加靶标单链DNA也会造成探针的切割。而FnCas12a等，只在加入靶标单链DNA时，发生探针的切割，是较好的候选Cas12a蛋白。
实施例3 Cas12a蛋白检测可探测双链DNA靶标
选用双链DNA(target-T1)作为靶标序列，测试不同Cas12a蛋白对其检测的响应值。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-T1-24-R(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，靶标双链DNA(target-T1，由引物target-T1-F与引物target-T1-R退火后获得)(0.01μM)，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min，然后98℃2min终止反应。
3、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图7所示，不同Cas12a对靶标检测效果不同。而LbCas12a等，只在加入靶标双链DNA时，发生探针的切割，是较好的候选Cas12a蛋白。
实施例4 FnCas12a与LbCas12a测试不同浓度靶标
选用target-T1作为靶标DNA，然后梯度稀释成不同浓度，测试FnCas12a与 LbCas12a对其响应灵敏度。为了增加灵敏度，加入了PCR扩增步骤。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-T1-24-R(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、PCR扩增(可选)：以含target-T1靶标的质粒(pUC18-T1)作为模板，梯度稀释，进行PCR反应。每个反应体系总体积为20μL，用0.25μM的M13F-47、M13R-48为引物(表4)，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，PCR扩增产物直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，FnCas12a或LbCas12a(0.25μM)，PCR产物1μL(或直接稀释成不同浓度的靶标DNA)，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图9所示，当直接加入测试靶标时，0.1nM浓度以上的靶标DNA都能够响应，而且浓度1nM以上时响应显著。如果结合PCR技术，即先通过PCR扩增目的片段再进行Cas12a切割反应，此时，响应灵敏度可以低至10aM。
实施例5 FnCas12a与LbCas12a测试单位点突变靶标
选用target-T1作为靶标，将其在PAM区与1-18位分别进行单位点突变，测试几种不同长度的crRNA，对野生型与单位点突变后的响应值。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-T1-24-R、T7-T1-15-R、T7-T1-16-R、T7-T1-17-R、T7-T1-18-R(表5)分别进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂 盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、PCR扩增：以含target-T1靶标的质粒(pUC18-T1)作为模板。每个反应体系总体积为20μL，用0.25μM的引物M13R-48与Target-T1-F的各个突变引物(表4)，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，FnCas12a或LbCas12a(0.25μM)，PCR产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图10所示，当靶标互补序列为24nt的crRNA(crRNA-24nt)时，8-18位的单位点突变与野生型差异不大，而PAM突变和1-7位点突变后，荧光值有明显的下降。当截短crRNA长度，配对靶标序列长度为18nt时，突变位置8-16nt的荧光值相比24nt有明显下降，当16nt或17nt时，突变后的靶标序列荧光值下降更加明显，而15nt时，靶标序列和突变的荧光值则都已很弱，但是针对其他的靶标序列时其强度可能仍会较高，可以用于检测。综合来看，15nt、16nt和17nt的crRNA对SNP的检测最为合适。
实施例6环境水体中大肠杆菌等微生物测试
选用大肠杆菌gyrB基因作为检测目标，间接测试水体中大肠杆菌等微生物的浓度。以大肠杆菌MG1655作为正对照，测定环境中的水(如污水与自来水)中微生物的含量。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-crRNA-gyrB(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，稀释为10 μM浓度并保存到-80℃冰箱中。
2、PCR扩增：正对照样品为大肠杆菌MG1655培养至OD 600约为0.5时，分别以10倍为梯度稀释后作为模板，样品为环境水(包括自来水和环境中的泥水)。每个反应体系总体积为20μL，用0.25μM的引物gyrB-F与gyrB-R(表4)，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，PCR产物直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，LbCas12a(0.25μM)，PCR产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图13所示，随着大肠杆菌MG1655浓度的减少，其荧光响应值逐渐减少。其中，样品2，4，5，6较明显的检测出了微生物。
实施例7人SNP测试
SNP测试选用了人SNP的5个位点，分别为rs5082，rs1467558，rs2952768，rs4363657，rs601338，测试HOLMES方法的可行性。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。使用RNA Clean&Concentrator TM-5(Zymo Research)纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、PCR扩增：反应体系总体积为20μL，用0.25μM的引物(表4)，1ng的人基因组(HEK293T)或直接刮取口腔上皮粘膜作为模板，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，直接用于Cas12a反应。(其中引物1-rs5082-F-T，2-rs1467558-F-T，3-rs2952768-R-C直接引入SNP的相应突变产物)
3、Cas12a反应：20μL反应体系中，加入相应的crRNA(1μM)，LbCas12a(0.5μM)，PCR产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)。37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图14，仅当crRNA对应相应的靶标序列时，才有较高的荧光响应值，如果有一个位点的突变，其响应值就会大大降低。通过荧光值得大小即可判断对应SNP的基因型，这些结果也得到了测序结果的确认。
实施例8癌症相关基因测试
选用了TP53基因作为测试基因，其中在人T24细胞中，TP53基因有一个无义突变，导致该基因失活。分别测试了该位点基因正常的细胞(HEK293T)，个人的基因测试，以及突变细胞T24。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-crRNA-34-TP53-T24-C-16nt与T7-crRNA-34-TP53-T24-G-16nt(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。使用RNA Clean&Concentrator TM-5(Zymo Research)纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、PCR扩增：反应体系总体积为20μL，用0.25μM的引物34-TP53-T24-F，34-TP53-T24-R(表4)，1ng的人基因组(HEK293T，T24)或直接刮取口腔上皮粘膜作为模板，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入相应的crRNA(1μM)，LbCas12a(0.5μM)，PCR产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)。37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nM，发射光556nM)。如图15，当该位点正常的TP53基因为模板检测到的crRNA-C的数值明显高于crRNA-G，而突变的细胞T24的crRNA-G则明显升 高。
实施例9人SNP测试(痛风相关基因)
SNP测试选用了人SNP的5个位点，这些与痛风的风险相关，分别为rs1014290，rs6449213，rs737267，rs1260326，rs642803，测试HOLMES方法。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。使用RNA Clean&ConcentratorTM-5(Zymo Research)纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度并保存到-80℃冰箱中。
2、PCR扩增：反应体系总体积为20μL，用0.25μM的引物(表4)，1ng的人基因组(HEK293T)或直接刮取口腔上皮粘膜作为模板，PCR反应用高保真酶KOD FX(ToYoBo)。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。PCR完成后，直接用于Cas12a反应。(其中引物1-rs5082-F-T，2-rs1467558-F-T，3-rs2952768-R-C直接引入SNP的相应突变产物)
3、Cas12a反应：20μL反应体系中，加入相应的crRNA(1μM)，LbCas12a(0.5μM)，PCR产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)。37℃反应15min，然后98℃2min终止反应。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。如图16，仅当crRNA对应相应的靶标序列时，才有较高的荧光响应值，如果有一个位点的突变，其响应值就会大大降低。通过荧光值得大小即可判断对应SNP的基因型，这些结果也得到了测序结果的确认。
实施例10试剂盒SNP测试志愿者临床样本(痛风相关基因)
将预混液加入到96孔板中，制备成试剂盒，然后加入21名志愿者的基因组DNA，测试rs1014290位点，该位点与痛风的风险相关。
1、试剂盒制备：首先，通过用T7-crRNA-F与合成的寡核苷酸(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen  Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。使用RNA Clean&ConcentratorTM-5(Zymo Research)纯化RNA，并用NanoDrop 2000C定量，稀释为10μM浓度。
2、96孔板PCR预混：在19μL体系中，加入PCR反应所需试剂，引物用41-rs1014290-F与41-rs1014290-R。
3、荧光检测96孔板预混：在19μL体系中，加入crRNA(1μM)，LbCas12a(0.5μM)，荧光探针(HEX-N12-BHQ1)(0.5μM)，加入到96孔板中。
4、PCR扩增：在PCR预混的96孔板中加入志愿者基因组DNA，然后进行PCR反应。PCR反应程序为，95℃2min，然后开始35个循环98℃10s，60℃15s，68℃10s。
5、Cas12a反应：取1μL PCR反应液，加入到已预混的荧光检测96孔板中，37℃反应15min，然后98℃2min终止反应。
6、荧光检测：用酶标仪检测(激发光535nm，发射光556nm)。如图17，由于基因型A:A的人群具有更高的痛风风险，因此除了5,7,9号志愿者外，其他人为A:G或G:G基因型，因此需更加注意痛风的风险。
实施例11用LAMP结合Cas蛋白检测环境水体中大肠杆菌等微生物
选用大肠杆菌gyrB基因作为检测目标，间接测试水体中是否存在大肠杆菌等微生物。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-crRNA-gyrB(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×Taq DNA polymerase反应缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，最终稀释为10μM浓度并保存到-80℃冰箱中备用。
2、LAMP扩增：以无菌水和含大肠杆菌的污染液体分别作为阴性对照和待检测样品。每个反应体系总体积为25μL，用引物1.6μM的LAMP-FIP与LAMP-BIP、0.2μM的LAMP-F3与LAMP-B3、0.4μM的LAMP-LoopF与LAMP-LoopB，LAMP反 应所用的试剂盒为
Figure PCTCN2018082769-appb-000002
LAMP Kit(NEB)。LAMP反应程序为，65℃30min。LAMP完成后，80℃10min灭火，然后直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，LAMP产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。结果如图19。
实施例12用LAMP扩增结合Cas蛋白检测SNP
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-crRNA-rs5082-T/T7-crRNA-rs5082-G/T7-crRNA-rs1467558-T/T7-crRNA-rs1467558-C(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×Taq DNA polymerase反应缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop2000C定量，最终稀释为10μM浓度并保存到-80℃冰箱中备用。
2、LAMP扩增：以人基因组HEK293T作为样品。每个反应体系总体积为25μL，用引物1.6μM的LAMP-FIP与LAMP-BIP、0.2μM的LAMP-F3与LAMP-B3、0.4μM的LAMP-LoopF与LAMP-LoopB，LAMP反应用
Figure PCTCN2018082769-appb-000003
LAMP Kit(NEB)。LAMP反应程序为，65℃30min。LAMP完成后，80℃10min灭火，然后直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，LAMP产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。结果如图20。
实施例13用RPA扩增结合Cas蛋白检测环境水体中大肠杆菌等微生物
选用大肠杆菌gyrB基因作为检测目标，间接测试水体中是否存在大肠杆菌等微生物。
1、crRNA制备：首先，通过用T7-crRNA-F与合成的寡核苷酸T7-crRNA-gyrB(表5)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成crRNA，并且反应在37℃下进行过夜(约16h)。然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop2000C定量，最终稀释为10μM浓度并保存到-80℃冰箱中备用。
2、RPA扩增：以无菌水和含大肠杆菌污染液体分别作为阴性对照和待检测样品。每个反应体系总体积为25μL，用引物0.5μM的RPA-gyrB-F(或RPA-gyrB-F2)与RPA-gyrB-R2，RPA反应用
Figure PCTCN2018082769-appb-000004
Basic试剂盒(TwistDX)。RPA反应程序为，37℃30min。RPA完成后，80℃10min灭火，然后直接用于Cas12a反应。
3、Cas12a反应：20μL反应体系中，加入步骤1中纯化的crRNA(0.5μM)，Cas12a(0.25μM)，RPA产物1μL，荧光探针(HEX-N12-BHQ1)(0.5μM)，缓冲液为NEB buffer 3.1，0.5μL RNA酶抑制剂。在37℃反应15min。
4、荧光检测：将灭活的20μL反应液加入96孔板中，然后用酶标仪检测(激发光535nm，发射光556nm)。结果如图21。
实施例14：Cas12b具有旁路切割活力
1、向导RNA(sgRNA)的制备
首先构建以pUC18为质粒骨架构建质粒pUC18-向导RNA-T1，该质粒在pUC18上插入了T7启动子及用于转录向导RNA的模板DNA序列(注：此模板转录出的向导RNA靶向一个在本研究中称为T1的序列)。方法是以pUC18质粒为模板，pUC18-1-F和pUC18-1-R为引物，先进行一轮PCR，T4DNA Ligase连接PCR产物，转化入DH10b中，经测序得到正确克隆，称之为pUC18-向导RNA-T1-pre。再以pUC18-向导RNA-T1-pre为模板，pUC18-2-F和pUC18-2-R为引物，进行第二轮PCR，同样地方法连接PCR产物并转化，最终得到测序正确的质粒pUC18-向导RNA-T1。
接着，以质粒pUC18-向导RNA-T1为模板，使用T7高产量转录试剂盒(Thermo)合成向导RNA，反应在37℃下过夜进行(12-16h)。
最后，在转录体系中加入DNaseⅠ(每50μL转录体系加2μL DNaseⅠ)，37℃水浴30min，消除质粒DNA，并使用RNA纯化与浓缩试剂盒纯化RNA，再用NanoDrop 2000C定量，稀释为10μM浓度并保存于-80℃冰箱中备用。
2、靶标DNA的制备
(1)若靶标DNA为单链，直接合成一条长66bp的寡核苷酸作target DNA(target-T1-R)，其中包含向导RNA所识别的20bp靶标序列(T1)。
(2)若靶标DNA为双链，直接合成两条互补的长66bp的寡核苷酸(target-T1-F；target-T1-R)，其中包含向导RNA所识别的20bp靶标序列(T1)。将两条寡核苷酸退火，得到短的target DNA。具体是，将配对的寡核苷酸(1μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为20μL，然后进行退火程序：初始在95℃变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。
3、Cas12b反应
(1)向导RNA退火：将向导RNA稀释至适当浓度(10μM)，置PCR仪中退火。退火程序：75℃变性5min，然后从75℃冷却至20℃，每分钟降低1℃。
(2)向导RNA与C2c1孵育：将退火完成的向导RNA与等摩尔浓度的C2c1混合，于30℃放置20-30min。
(3)Cas12b反应：在20μL反应体系中，加入步骤(2)孵育的向导RNA与C2c1的混合物(两者终浓度均为250μM或500μM)，target DNA(终浓度50nM)，FAM标记的寡核苷酸(target-DNMT1-3-R-FAM-5’)或荧光淬灭探针(HEX-N12-BHQ1，终浓度500nM)，以及2μL的10×NEB Buffer 3.1和0.5μL的RNA酶抑制剂(40U/μL)。混匀后，于48℃反应30min。之后，置PCR仪中98℃加热5min灭活。
4、尿素变性凝胶电泳法检测Cas12b的trans切割(反式切割)活力
将灭活的20μL反应液利用尿素变性凝胶电泳法进行电泳分离，然后使用荧光成像系统ImageQuant LAS 4000mini(GE Healthcare)进行成像。结果如图22。
5、荧光酶标仪法检测Cas12b的trans切割(反式切割)活力
将灭活的20μL反应液加入96孔板中，用酶标仪检测(激发光535nm，发射光556nm)。结果如图23。
实施例15：Cas12b的trans反应(反式切割)的灵敏度测试
通过对荧光探针(HEX-N12-BHQ1)被激发的荧光强度进行检测，确定Cas12b行使反式切割活力需要的靶标DNA浓度，即Cas12b反式切割反应的灵敏度。
1、向导RNA的制备
首先，以pUC18-向导RNA-T1为模板，向导RNA-DNMT1-3-F和向导RNA-DNMT1-3-R为引物，通过PCR将向导RNA中靶向T1这一target DNA的20个碱基替换为靶向DNMT1-3的向导RNA，得到另一质粒pUC18-向导RNA-DNMT1-3。
然后，以质粒pUC18-向导RNA-DNMT1-3为模板，使用T7高产量转录试剂盒(Thermo)合成向导RNA，反应在37℃下过夜进行(12-16h)。
最后，在转录体系中加入DNaseⅠ(每50μL转录体系加2μL DNaseⅠ)，37℃水浴30min，消除质粒DNA，并使用RNA纯化与浓缩试剂盒纯化RNA，再用NanoDrop 2000C定量，于-80℃冰箱中保存备用。
2、靶标DNA的制备
对于靶标DNA，第一种是直接加入Cas12b的反应体系，不经过扩增。方法如下：
(1)若靶标DNA为单链，直接合成一条长50bp的寡核苷酸作target DNA(DNMT1-3(TTC PAM)-R)，其中包含向导RNA所识别的20bp靶标序列(DNMT1-3)。
(2)若靶标DNA为双链，直接合成两条互补的长50bp的寡核苷酸(DNMT1-3(TTC PAM)-F；DNMT1-3(TTC PAM)-R)，其中包含向导RNA所识别的20bp靶标序列(DNMT1-3)。将两条寡核苷酸退火，得到短的target DNA。具体是，将配对的寡核苷酸(2μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为20μL，然后进行退火程序：初始在95℃变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。
(3)将单链或双链靶标DNA梯度稀释为2μM，0.2μM，0.02μM，0.002μM，0.0002μM备用。
第二种是将含靶标序列(DNMT1-3)的片段插入一质粒载体中，通过LAMP反应扩增。
(1)使用Transgen公司的pEasy-Blunt Zero Cloning Kit，将含靶标序列(DNMT1-3)片段插入pEasy-Blunt Zero Cloning Vector，经测序验证后获得正确的克隆。
(2)LAMP扩展反应
以上述质粒作为模板，进行LAMP扩增反应，模板分别加入0，1nM，0.1nM，10倍梯度稀释至10 -11nM。每个反应体系总体积为25μL，用引物1.6μM的LAMP-DNM-FIP与LAMP-DNM-BIP、0.2μM的LAMP-DNM-F3与LAMP-DNM-B3、0.4μM的LAMP-DNM-LoopF与LAMP-DNM-LoopB，LAMP反应所用的试剂盒为
Figure PCTCN2018082769-appb-000005
LAMP Kit(NEB)。LAMP反应程序为，65℃30min。LAMP完成后，80℃10min灭火，然后直接用于Cas12b反应。
3、Cas12b反应
(1)向导RNA退火：将向导RNA稀释至适当浓度(5μM)，置PCR仪中退火。退火程序：75℃变性5min，然后从75℃冷却至20℃，每分钟降低1℃。
(2)向导RNA与Cas12b孵育：将退火完成的向导RNA与等摩尔浓度的Cas12b混合，于30℃放置20-30min。
(3)Cas12b反应：在20μL反应体系中，加入步骤(2)孵育的向导RNA与Cas12b的混合物(两者终浓度均为250μM)，1μL的target DNA或1μL的LAMP产物，荧光探针(HEX-N12-BHQ1)(终浓度500nM)，以及2μL的10×NEB Buffer 3.1和0.5μL的RNA酶抑制剂(40U/μL)。混匀后，于48℃反应30min。之后，置PCR仪中98℃加热5min灭火。
4、荧光酶标仪法检测Cas12b的trans切割(反式切割)活力
将灭活的20μL反应液加入96孔板中，用酶标仪检测(激发光535nm，发射光556nm)。结合了LAMP扩增之后，Cas12b可以针对低至10aM浓度的靶标DNA产生很显著的旁路单链DNA反式切割活力。结果如图24。
Cas12a切割靶标单链DNA的顺式切割特性：
首先，为了测试Cas12a的单链DNA切割特征，设计了几个靶向短的单链DNA(DNMT1-3)的crRNA(表1)，其在3’末端用5(6)-羧基荧光素(FAM)标记。在FnCas12a切割后，通过变性尿素聚丙烯酰胺凝胶电泳(尿素PAGE)分析反应产物。发现通过Cas12a的单链DNA切割是程序化的，即切割位点为从第一个与crRNA引导序列配对的靶标序列的3’端碱基开始向5’端计数的靶标序列第22个碱基附近(第21至第23个碱基)，如图1A和1C所示。Cas12a的双链DNA需要PAM序列，而单链DNA切割不需要PAM序列(图1A，1B与图2)，这与Cas9介导的单链DNA切割相似。然而，Cas12a介导的单链DNA切割活性依赖于crRNA中的茎环结构，如图1A所示，而Cas9仍然表现出对仅具有20-nt互补RNA序列的单链DNA的仍有弱切割活性。crRNA的茎环结构对于稳定Cas12a的结构是重要的，其为crRNA环结构对Cas12a的单链DNA切割的必要性的原因。进一步测试Cas12a的单链DNA切割位点是否可以通过更短的引导序列crRNA，从而使得切割在识别位点之外。在引导序列长度为16nt，18nt和20nt时，所有这些crRNA都导致在靠近第22个碱基处通过Cpf1切割，如图1B与1D所示，意味着切割位点是识别 位点之外的4nt，2nt或0nt。接着，测试了Cas12a对不同底物的切割效率，分别用了双链DNA和单链DNA底物，如图1F所示。类似于Cas9切割情况，单链DNA切割比双链DNA切割更慢，如图1E至1G所示。这些结果表明Cas12a的单链DNA识别与切割的机制可能不同于双链DNA，是一种效率较低的不依赖于PAM的识别切割方式；PAM序列为Cas12a对靶标双链DNA的识别和/或切割起到加速的作用。
Cas12a切割单链DNA的反式切割特性：
当靶标单链DNA在3’末端进行标记时，Cas12a切割第22个碱基附近，如图1所示。然而，当标记在5’末端时，没有观察到预测大小的切割产物条带，但是产生短(<6nt)FAM标记的产物，如图3B所示。通过详细的实验，一旦三元复合体Cas12a/crRNA/靶标单链DNA形成，5’-末端标记的靶标单链DNA(DNMT1-3)(表1)被切割并产生短FAM标记的产物，如图3C所示。此外，三元复合体还切割任何其他反应体系中与crRNA没有互补性序列的单链DNA(即旁路单链DNA)，如图3C和图3D所示。将这种切割现象为反式切割，区别于可程序化的顺式切割。当靶标单链DNA在3’-末端标记时，也观察到了反式切割，但留下了许多顺式切割产物，如图3B所示，这可能是由于Cas12a/crRNA/靶标单链DNA形成了复合物，并保护了靶标单链DNA，以使其标记的3’-末端免于暴露于三元复合物的核酸酶活性位点，这些切割过程可以用图3A显示。
除了上面测试的FnCas12a，还测试了来自其他物种来源的9种Cas12a(表2和图4A)。除了Lb4Cas12a以外，所有的Cas12a都对质粒DNA具有较好的核酸内切酶活性(如图4B所示)，并且所有的Cas12a三元复合体都显示出对单链的顺式和反式切割活性(如图4C和4D所示)。这说明Cas12a针对单链DNA的顺式和反式活力是普遍现象。
Cas12a切割单链DNA的顺式和反式关键位点与机理
为了测定Cas12a中有关对单链DNA顺式和反式活性的关键氨基酸残基，突变了Cas12a的几个候选残基进行活力测试。首先，纯化并测试FnCas12a(H843A，K852A和K869A)的三个单氨基酸突变体，其残基与RNase活性相关。对于单链DNA的反式活性研究结果显示，野生型FnCas12a和三个突变体在单链DNA顺式和反式切割活力上没有发现明显的差异，如图5A和5C所示。
接着，当FnCas12a中的核酸内切酶活性位点突变时，即RuvC结构域(D917A，E1006A或D1255A)与Nuc结构域(R1218A)位点，这些突变Cas12a的单链DNA顺式和反式切割活性都受到了影响，如图5B和5D所示。这些结果表明Cas12a针对靶标双链DNA切割的关键位点与单链DNA顺式和反式切割活性密切相关。
最近对Cas12b(即C2c1)的结构研究(包括与延伸的靶DNA或延伸的非靶DNA复合体)显示两条链均位于RuvC口袋内，如图6A和6B所示。通过比较Cas12b(即C2c1)和Cas12a的核酸内切酶催化残基，这些位点最可能在Cas12b(即C2c1)和Cas12a的切割和功能中发挥类似的作用。体外单氨基酸突变实验的结果表明，与上述假设是一致的，也即是说Cas12a很可能通过只通过一个RuvC催化口袋切割两条链。
Cas12a复合体的反式切割活性：在Cas12b(即C2c1)复合体与额外单链DNA的结构中，序列非依赖性单链DNA也位于催化口袋的表面，如图6C所示，这类似于Cas12a中的旁路单链DNA底物。结合单个氨基酸突变实验，提出标靶DNA，非靶标DNA和旁路单链DNA都在Cas12a中的单个RuvC口袋切割，如图6D，6E和6F所示。三元Cas12a复合体具有旁路单链DNA反式切割活性，而单体或二元复合体没有旁路单链DNA反式切割活性的原因可以通过比较单体、二元和三元复合体的结构来解释。单体Cas12a结构是无序的，二元复合体Cas12a/crRNA是三角形结构，如图6G所示，而三元复合物Cas12a/crRNA/靶标DNA转变为双叶结构，从而暴露出催化口袋，以实现旁路单链DNA的反式切割(如图6H所示)。
核酸探测方法的建立
利用Cas12a的特性，开发了特异性检测核酸分子的方法，称之为HOLMES(one HOur Low-cost Multipurpose Efficient Simple assay)。正如该技术的名称一样，其特点是一小时、低成本、多用途、高效率、很简便的测试法。
在整个反应体系中，可分为两个大的步骤，一个是对模板核酸的扩增，另一个是Cas12a蛋白的特异性核酸检测。这里，对于核酸的扩增运用了PCR的方法，但实际上，任何的扩增方法都可结合第二步的核酸检测，例如等温扩增方法RPA等。初始的核酸不限于双链DNA，也可以是单链DNA；即使是RNA，也可以通过反转录后实现检测，因此本方法适用于多种类型的核酸分子。对于核酸检测阶段，其中3个组分是实验的关键，分别为Cas12a，crRNA和核酸探针。除了实施例中提到的10种Cas12a(这10种蛋白是随机选择的)，其他Cas12a蛋白同样适用于该方法。此外，其它类型的Cas蛋白(如C2c1蛋白)也适用于本发明保护的范畴：根据实验结果显示，Alicyclobacillus acidoterrestris Cas12b(即C2c1)也具有与Cas12a类似的旁路单链DNA反式切割活力，其与crRNA/靶标DNA的复合物也可以切割旁路单链DNA。
对于作为引导作用的crRNA，经过人工修饰等改造后，在体系中会更加稳定。在核酸探针的选择上，本发明选用了HEX和BHQ1标记的短单链DNA，其他可检测的任何标记方式理论上都是适用的，只要该核酸探针被切割后产生可检测的差异。或者，核酸探针也可以设计成可以跟化合物结合后发荧光，从而探测是否该探针 被切断。
此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
表1 Cas12a特性实验相关切割底物序列
Figure PCTCN2018082769-appb-000006
Figure PCTCN2018082769-appb-000007
表2.Cas12a蛋白和Cas12b(即C2c1)蛋白的名称与GI号
Name GI number Species
FnCas12a 489130501 Francisella tularensis
AsCas12a 545612232 Acidaminococcus sp.BV3L6
LbCas12a 917059416 Lachnospiraceae bacterium ND2006
Lb5Cas12a 652820612 Lachnospiraceae bacterium NC2008
HkCas12a 491540987 Helcococcus kunzii ATCC 51366
OsCas12a 909652572 Oribacterium sp.NK2B42
TsCas12a 972924080 Thiomicrospira sp.XS5
BbCas12a 987324269 Bacteroidales bacterium KA00251
BoCas12a 496509559 Bacteroidetes oral taxon 274str.F0058
Lb4Cas12a 769130406 Lachnospiraceae bacterium MC2017
C2c1 1076761101 Alicyclobacillus acidoterrestris
表3 质粒信息
Figure PCTCN2018082769-appb-000008
Figure PCTCN2018082769-appb-000009
表4 HOLMES方法测试中用到的引物
Figure PCTCN2018082769-appb-000010
Figure PCTCN2018082769-appb-000011
Figure PCTCN2018082769-appb-000012
Figure PCTCN2018082769-appb-000013
表5 用于转录crRNA的模板序列
Figure PCTCN2018082769-appb-000014
Figure PCTCN2018082769-appb-000015
Figure PCTCN2018082769-appb-000016
Figure PCTCN2018082769-appb-000017
LAMP扩增DNA结合Cas12a检测所用到的引物：
表6 扩增gyrB-1的引物
Figure PCTCN2018082769-appb-000018
表7 扩增gyrB-2的引物
Figure PCTCN2018082769-appb-000019
Figure PCTCN2018082769-appb-000020
表8 扩增rs1467558位点的引物
Figure PCTCN2018082769-appb-000021
表9 扩增rs5082位点的引物
Figure PCTCN2018082769-appb-000022
表10 RPA扩增结合Cas12检测用到的引物
名称 序列 SEQ ID No.:
RPA-gyrB-1-F ATATGAAATTCTGGCGAAACGTCTGCGTGAGTTG 154
RPA-gyrB-2-F AAACGTCTGCGTGAGTTGTCGTTCCTCAACTCC 155
RPA-gyrB-R ACTTCGACGCCAATACCGTCTTTTTCAGTGGAG 156
表11 测定Cas12b具有trans切割活性用到的引物：
Figure PCTCN2018082769-appb-000023
表12 Cas12b的trans反应的灵敏度测试用到的引物：
Figure PCTCN2018082769-appb-000024
Figure PCTCN2018082769-appb-000025
表13 本发明涉及到的其它序列
Figure PCTCN2018082769-appb-000026
Figure PCTCN2018082769-appb-000027
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (21)

  1. 一种靶标核酸分子的检测方法，其特征在于，向含有待检靶标核酸分子的体系中加入向导RNA、Cas蛋白、核酸探针、缓冲液，然后对核酸探针进行检测。
  2. 根据权利要求1所述的靶标核酸分子的检测方法，其特征在于，所述Cas蛋白为Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白；
    所述的Cas12a优选为FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a中一种；所述的Cas12a优选为LbCas12a。
  3. 根据权利要求1所述的靶标核酸分子的检测方法，其特征在于，向导RNA是指引导Cas蛋白特异性结合靶标DNA的RNA。
  4. 根据权利要求1所述的靶标核酸分子的检测方法，其特征在于，所述核酸探针为单链DNA；所述的单链DNA优选为荧光标记的单链DNA；所述的单链DNA优选为在5’端标记荧光基团HEX并在3’端标记淬灭基团BHQ1后的荧光探针；优选地，
    所述核酸探针的检测方法优选为荧光检测法；所述的荧光检测法优选为使用酶标仪或者荧光分光光度计进行检测的方法。
  5. 根据权利要求1至4中任意一项所述的靶标核酸分子的检测方法，其特征在于，待检靶标核酸分子的反应体系中的待检靶标核酸分子通过扩增得到。
  6. 根据权利要求5所述的靶标核酸分子的检测方法，其特征在于，该检测方法可检测病原微生物、基因突变或特异靶标DNA。
  7. 如权利要求1所述的方法，其特征在于，所述的Cas蛋白包括Cas12b(即C2c1)。
  8. 一种Cas蛋白在靶标核酸分子的检测方法中的用途。
  9. 根据权利要求8所述的用途，其特征在于，靶标DNA、向导RNA和Cas蛋白形成三元复合体时，该复合物会切割体系中其它的单链DNA分子；优选地，向导RNA是指引导Cas蛋白特异性结合靶标DNA的RNA。
  10. 一种靶标核酸分子的检测试剂盒，其特征在于，包括向导RNA、Cas蛋白、核酸探针。
  11. 一种用于检测靶标核酸分子的检测体系，其特征在于，该体系包含：
    (a)Cas蛋白，所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白；
    (b)向导RNA，所述向导RNA引导Cas蛋白特异性结合于靶标核酸分子；和
    (c)核酸探针，所述核酸探针为单链DNA；
    其中，所述的靶标核酸分子为靶标DNA。
  12. 如权利要求11所述的检测体系，其特征在于，所述的具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白选自下组：Cas12b(即C2c1)。
  13. 如权利要求11所述的检测体系，其特征在于，所述的核酸探针包括带有可检测标记的单链DNA。
  14. 一种用于检测靶标核酸分子的试剂盒，其特征在于，所述试剂盒包括：
    i)第一容器以及位于第一容器内的Cas蛋白，所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白；
    ii)任选的第二容器以及位于第二容器内的向导RNA，所述向导RNA引导所述Cas蛋白特异性结合于靶标核酸分子；
    iii)第三容器以及位于第三容器内的核酸探针；
    iv)任选的第四容器以及位于第四容器内的缓冲液；
    其中，所述的靶标核酸分子为靶标DNA。
  15. 一种检测样本中是否存在靶标核酸分子的方法，其特征在于，包括以下步骤：
    (a)提供权利要求11所述的用于检测靶标核酸分子的检测体系，并且所述的检测体系还具有待检测的样本；和
    (b)检测所述检测体系中的核酸探针是否被Cas蛋白进行切割，所述的切割为旁路单链DNA的反式切割；
    其中，所述核酸探针被Cas蛋白切割，则表示所述样本中存在靶标核酸分子；而所述核酸探针不被Cas蛋白切割，则表示所述样本中不存在靶标核酸分子。
  16. 如权利要求15所述的方法，其特征在于，所述核酸扩增的方法选自下组：PCR扩增、LAMP扩增、RPA扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA,SPIA,NEAR,TMA和SMAP2。
  17. 如权利要求15所述的方法，其特征在于，当所述的靶位点的上下游(-20nt至+20nt范围内，较佳地-15nt至+15nt范围内，更佳地-10nt至+10nt范围内)缺乏PAM序列时，采用引入PAM的引物进行核酸扩增。
  18. 如权利要求17所述的方法，其特征在于，所述的引入PAM的引物具有从5’-3’的式I结构：
    P1-P2-P3  (I)
    式中，
    P1为位于5’端的与靶标核酸分子的序列互补或非互补的5’区段序列；
    P2为PAM序列；
    P3为位于3’端的与靶标核酸分子的序列互补3’区段序列。
  19. 如权利要求15所述的方法，其特征在于，当所述的靶位点的上下游(-20nt至+20nt范围内，较佳地-15nt至+15nt范围内，更佳地-10nt至+10nt范围内)含有PAM序列时，则可采用含有或不含有PAM序列的引物，并且扩增出的扩增产物含有所述PAM序列。
  20. 一种Cas蛋白的用途，其特征在于，用于制备基于旁路单链DNA切割检测靶标核酸分子的检测试剂或试剂盒，其中所述Cas蛋白是Cas12a或者具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白。
  21. 如权利要求20所述的用途，其特征在于，所述的具有与Cas12a的旁路单链DNA切割活性类似的Cas蛋白选自下组：Cas12b(or C2c1)。
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