CN118272491A

CN118272491A - A method for detecting crRNA to determine whether a target nucleic acid molecule exists and related products

Info

Publication number: CN118272491A
Application number: CN202211728536.6A
Authority: CN
Inventors: 刘涛; 陈敏洁; 王金
Original assignee: Shanghai Tolo Biotechnology Co ltd
Current assignee: Shanghai Tolo Biotechnology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2024-07-02

Abstract

披露了一种利用Cas酶的核酸检测方法，提供了检测crRNA以判断靶标核酸分子是否存在的方法及相关产品，具体地，提供了用于检测靶标核酸分子例如靶标RNA分子的检测体系，包括第I体系和第II体系，所述第I体系包括：a)Cas蛋白；b)Cas蛋白顺式切割底物或Cas蛋白顺式切割报告分子；还可以进一步包括：c)Cas蛋白反式切割报告分子；所述第II体系包括根据靶标核酸分子例如靶标RNA分子制备完整或部分的crRNA的物质。通过将靶标核酸分子例如靶标RNA分子实时制备成crRNA，可以实现在Cas蛋白、Cas蛋白顺式切割底物存在的情况下检测靶标核酸分子，尤其是直接检测样本中的靶标RNA分子。A nucleic acid detection method using Cas enzyme is disclosed, and a method and related products for detecting crRNA to determine whether a target nucleic acid molecule exists are provided. Specifically, a detection system for detecting a target nucleic acid molecule such as a target RNA molecule is provided, including a first system and a second system, wherein the first system includes: a) Cas protein; b) Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter molecule; and may further include: c) Cas protein trans-cleavage reporter molecule; and the second system includes a substance for preparing complete or partial crRNA according to a target nucleic acid molecule such as a target RNA molecule. By preparing a target nucleic acid molecule such as a target RNA molecule into crRNA in real time, it is possible to detect a target nucleic acid molecule in the presence of a Cas protein or a Cas protein cis-cleavage substrate, especially directly detecting a target RNA molecule in a sample.

Description

Method for detecting crRNA to judge whether target nucleic acid molecule exists or not and related products

Technical Field

The invention relates to the field of biotechnology, in particular to a method for detecting crRNA to judge whether a nucleic acid target nucleic acid molecule exists or not and a related product, and in particular relates to a method for detecting the target nucleic acid molecule by using Cas protein and a related product.

Background

CRISPR is a microbiologically adaptive immune system that can be found in about 50% of prokaryotes and about 90% of archaebacteria. This immune system displays an endonuclease activity directed specifically against invasion from phage, transposon or plasmid. Currently, CRISPR-Cas systems have been applied to various applications, such as gene editing, imaging and detection of nucleic acids.

CRISPR-Cas systems can be used to detect RNA, cas13a first demonstrated to be able to cleave specific RNA sequences in bacterial cells and after cleavage to exert its trans-cleavage activity, continuing to cleave other non-target RNAs. The crRNA-target RNA duplex binds into one positively charged central channel of nuclease lobes (NUCs) in LbuCas a, and once the target RNA is bound, a significant conformational change occurs between LbuCas13a and the crRNA. This crRNA-target RNA duplex formation promotes the movement of the HEPN1 domain of LbuCas a towards the HEPN2 domain, thereby activating the HEPN catalytic site of LbuCas a, and subsequently LbuCas a cleaves single stranded target RNA and other RNAs in a non-specific manner. The first one used to detect RNA was a detection platform (DOI: 10.1126/science. Aam9321) named SHERLOCK (SPECIFIC HIGH-SENSITIVITY ENZYMATIC REPORTER UNLOCKING).

The CRISPR-Cas12 protein is found to have high-efficiency trans-cleavage activity for the first time by the team of King golden doctor of the original Chinese academy of sciences, and a Fulmos/HOLMES nucleic acid rapid detection platform technology (one-HOur Low-cost Multipurpose HIGHLY EFFICIENT SYSTEM) is developed based on the unique advantages of the enzyme, and is mainly used for detecting DNA. HOLMES can also detect RNA indirectly, requiring reverse transcription of the RNA into cDNA.

Disclosure of Invention

The present invention is directed to a method for detecting crRNA to determine the presence or absence of a target nucleic acid molecule.

The first aspect of the present invention provides a target nucleic acid molecule detection system comprising a system I, said system I being a crRNA detection system, said system I comprising:

a) Cas protein;

b) A Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter;

wherein the Cas protein cis-cleaves a substrate or Cas protein cis-cleaves a reporter (comprising a sequence identical or complementary to the target nucleic acid molecule sequence;

The Cas protein cis-cleavage substrate is not the target nucleic acid molecule from which the crRNA was prepared.

In another preferred embodiment, the target nucleic acid molecule comprises a target RNA molecule, a target DNA molecule.

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule and the Cas protein cis-cleavage substrate comprises a sequence identical or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system further comprises a second system; the system II is a crRNA synthesis system;

the system II is an RNA amplification system, the system II comprising:

d) A reverse transcriptase;

e) A primer, the primer structure is as follows:

primer 1:5'-Z1-Z2-Z3-3';

primer 2:5-Z4-3;

f) An RNA polymerase;

wherein, Z1 is an RNA polymerase specific recognition sequence;

Z2 is the complete or partial DR sequence;

z3 is a sequence capable of hybridizing to a portion of the target RNA molecule or its complement;

z4 is a sequence capable of hybridizing to a portion of the target RNA molecule or its complement;

preferably, when the reverse transcriptase has no function of digesting RNA, the II system further comprises an RNase H enzyme.

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule and the Cas protein cis-cleavage substrate comprises a sequence identical or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system further comprises a II 'system and a III' system; the II 'system and the III' system form a crRNA synthesis system, and the crRNA synthesis system is selected from group A 'or group B';

Group a' is as follows:

The system II 'is an RNA reverse transcription system, and the system II' comprises:

d') a reverse transcriptase;

h') a primer, wherein the primer structure is as follows: 5' -Z4' -3';

The Z4' is a sequence capable of hybridizing to a portion of the target RNA molecule;

The III 'system is a cDNA transcription and amplification system, the III' system comprising:

e') a primer, wherein the primer structure is as follows:

5' -Z1' -Z2' -Z3' -3'; and

F') RNA polymerase;

wherein, Z1' is an RNA polymerase specific recognition sequence;

Z2' is the complete or partial DR sequence;

z3' is a sequence capable of hybridizing to a portion of the target RNA molecule or its complement;

Preferably, when the reverse transcriptase has no function of digesting RNA, the II' system further comprises an RNase H enzyme;

group B' is as follows:

wherein the system II 'is an RNA reverse transcription system, the system II' comprising:

d') a reverse transcriptase;

h') a primer, wherein the primer structure is as follows: 5' -Z1' -Z2' -Z3' -3';

e') a primer, wherein the primer structure is as follows: 5' -Z4' -3';

f ") RNA polymerase;

wherein, Z1' is an RNA polymerase specific recognition sequence;

Z2' is the complete or partial DR sequence;

z3' is a sequence capable of hybridizing to a portion of the target RNA molecule;

z4' is a sequence capable of hybridizing to a portion of the target RNA molecule or its complement;

preferably, when the reverse transcriptase has no function of digesting RNA, the II' system further comprises an RNase H enzyme.

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule and the Cas protein cis-cleavage substrate comprises a sequence identical or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system further comprises a II ' system, a III ' system and an IV ' system; the system II, the system III and the system IV form a crRNA synthesis system, and are selected from the group A or the group B;

Group a "group:

The system II is an RNA reverse transcription system, and the system II comprises:

d') reverse transcriptase;

h ") primer, the primer structure is: 5' -Z4' -3';

said Z4 "is a sequence capable of hybridizing to a portion of said target RNA molecule;

The III "system is a cDNA amplification system, the III" system comprising:

e ") a primer, the primer structure being: 5' -Z1' -Z2' -Z3' -3'; and

F ") a DNA polymerase;

wherein, the Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

z3' is a sequence that hybridizes to the target RNA molecule or a portion of its complement;

the IV "system is a DNA transcription system, the IV" system comprising:

g ") RNA polymerase;

Preferably, when the reverse transcriptase has no function of digesting RNA, the II "system further comprises an RNase H enzyme;

Group B:

d') reverse transcriptase;

h ") primer, the primer structure is: 5' -Z1' -Z2' -Z3' -3';

wherein, the Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

The III "system is a cDNA amplification system, the III" system comprising:

e ") a primer, the primer structure being:

5'-Z4"-3', Z4" is a sequence that hybridizes to a portion of the target RNA molecule or its complement;

And

F ") a DNA polymerase;

the IV "system is a DNA transcription system, the IV" system comprising:

g ") RNA polymerase;

preferably, when the reverse transcriptase has no function of digesting RNA, the II "system further comprises an RNase H enzyme.

In another preferred embodiment, the target nucleic acid molecule is a target DNA molecule, and the Cas protein cis-cleavage substrate comprises a sequence identical or complementary to the target DNA molecule; the target nucleic acid molecule detection system further comprises a III '"system and an IV'" system; the III 'system and the IV' system form a crRNA synthesis system; wherein the method comprises the steps of

The III '"system is a DNA amplification system, the III'" system comprising:

e') primer, the primer structure is as follows:

primer 1:5 '-Z1' -Z2 ',' Z3 '-3',

Primer 2:5' -Z4 ' -3';

And

F' ") DNA polymerase;

wherein, the Z1' "is an RNA polymerase specific recognition sequence;

Z2' "is the complete or partial DR sequence;

Z3' "is a sequence that hybridizes to the target DNA molecule or a portion of its complement;

Z4' "is a sequence capable of hybridizing to a portion of said target DNA molecule or a complement thereof;

the IV-th '"system is a DNA transcription system, the IV-th'" system comprising:

g' ") RNA polymerase.

In another preferred embodiment, the system I further comprises a Cas protein cis-cleavage reporter, the Cas protein cis-cleavage substrate being part of the Cas protein cis-cleavage reporter; the Cas protein cis-cleavage substrate is selected from the group consisting of: single-stranded DNA, double-stranded DNA, single-stranded RNA, or a combination thereof.

In another preferred embodiment, the Cas protein cis-cleavage reporter is selected from the group consisting of: gold nanoparticle-based reporter molecules, fluorophore-based reporter molecules, fluorescence polarization-based reporter molecules, colloidal phase change/dispersion-based reporter molecules, electrochemical signal-based reporter molecules, semiconductor signal-based reporter molecules, color-based reporter molecules.

In another preferred embodiment, the Cas protein cis-cleavage reporter is detectably labeled.

In another preferred embodiment, the detectable label comprises a fluorescent group, a quenching group.

In another preferred embodiment, the fluorophore is selected from the group consisting of: FAM, HEX, cy3, cy5, cy5.5, cy7, ROX, VIC, JOE, TET, texas Red, FITC, LC RED640, RB200, or a combination thereof.

In another preferred embodiment, the quenching group is selected from the group consisting of: TAMARA, BHQ1, BHQ2, BHQ3, DABSYL, dabcyl, eclipse, or combinations thereof.

In another preferred embodiment, the fluorophore and the quencher are each independently located at the 5 'end, 3' end, and middle of the cis-cleavage substrate of the Cas protein.

In another preferred embodiment, the Cas protein cis-cleavage substrate is part of the Cas protein cis-cleavage reporter.

In another preferred embodiment, the Cas protein cis-cleavage reporter has a length of 3-300nt, preferably 5-100nt, more preferably 5-50nt, most preferably 5-15nt.

In another preferred embodiment, the Cas protein cis-cleavage reporter comprises single stranded DNA.

In another preferred embodiment, the Cas protein cis-cleavage reporter comprises single-stranded DNA with a detectable label.

In another preferred embodiment, the single-stranded DNA is fluorescent and biotin-labeled single-stranded DNA.

In another preferred embodiment, the single-stranded DNA is a fluorescent-labeled single-stranded DNA.

In another preferred embodiment, the target nucleic acid molecule comprises a DNA or RNA target derived from a member selected from the group consisting of: plants, animals, insects, microorganisms, viruses, or combinations thereof.

In another preferred embodiment, the target nucleic acid molecule is selected from the group consisting of: nucleic acid molecules of pathogenic microorganisms, genetically mutated nucleic acid molecules, and specific target nucleic acid molecules, or combinations thereof.

In another preferred embodiment, the pathogenic microorganism is selected from the group consisting of: viruses, bacteria, chlamydia, mycoplasma, or combinations thereof.

In another preferred embodiment, the virus is selected from the group consisting of: plant viruses, animal viruses, or combinations thereof.

In another preferred embodiment, the virus is selected from the group consisting of: coronavirus, influenza virus, HIV, hepatitis virus, parainfluenza virus.

In another preferred embodiment, the virus is a coronavirus.

In another preferred embodiment, the virus is selected from the group consisting of: SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV, or combinations thereof.

In another preferred embodiment, the target nucleic acid molecule comprises a wild-type or mutant target nucleic acid molecule.

In another preferred embodiment, the RNA polymerase is selected from the group consisting of: t7RNA polymerase, T3RNA polymerase, SP6RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, and single subunit RNA polymerase or combinations thereof.

In another preferred embodiment, the detecting includes: qualitative or quantitative detection.

In another preferred embodiment, the detection system further comprises (e) a buffer.

In another preferred embodiment, the detection system further comprises a target nucleic acid molecule.

In another preferred embodiment, the detection system further comprises reagents for a target nucleic acid molecule amplification reaction.

In another preferred embodiment, the target nucleic acid molecule amplification reaction is selected from the group consisting of: PCR (Polymerase Chain Reaction Amplification ), LAMP (Loop-mediated isothermal Amplification, loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification ), LCR (LIGASE CHAIN reactions, Ligase chain reaction), bDNA (branched DNA Amplification ), NASBA (Nuclear Acid Sequence-Based Amplification, nucleic acid sequence dependent Amplification), SDA (STRAND DISPLACEMENT Amplification ), TMA (Transcription-Mediated Amplification, transcription-mediated Amplification), RCA (Rolling Circle Amplification ), HDA (Helicase-DEPENDENT AMPLIFICATION, helicase dependent amplification), SPIA (SINGLE PRIMER Isothermal Amplification ), NEAR (Nicking Enzyme Amplification Reaction, nicking enzyme amplification reaction), SMAP2 (Smart Amplification Process version 2, Intelligent amplification method version 2), CPA (Cross Priming Amplification, cross primer amplification), MDA (Multiple Displacement Amplification ), RAM (Ramification), cHDA (circular Helicase-DEPENDENT AMPLIFICATION, helicase dependent circular amplification), SMART (SIGNAL MEDIATED Amplification of RNA Technology, Signal-mediated amplification of RNA), 3SR (Self-Sustained Sequence Replication, autonomous sequence replication system), GEAR (Genome Exponential Amplification Reaction ), IMDA (Isothermal Multiple Displacement Amplification, isothermal multiplex displacement amplification), ERA (Enzymatic Recombinase Amplification, enzymatic recombinant isothermal amplification) or a combination thereof.

In another preferred embodiment, the concentration of the target nucleic acid molecule in the detection system is 1X 10 ^-9nM-1×10³ nM; preferably 1X 10 ^-8nM-1×10² nM.

In another preferred embodiment, the concentration of the target nucleic acid molecule in the detection system is 1-100 copies/microliter or 1-1X 10 ¹⁵ copies/microliter, preferably 1-10 copies/microliter, more preferably 1-5 copies/microliter.

In another preferred embodiment, when the Cas protein cis-cleavage substrate is double-stranded DNA, the Cas protein cis-cleavage substrate contains a PAM sequence.

In another preferred embodiment, when the Cas protein cis-cleavage substrate is single-stranded DNA, the Cas protein cis-cleavage substrate does not contain a PAM sequence.

In another preferred embodiment, the length of the targeting sequence of crRNA synthesized by the RNA polymerase is 18-120nt, preferably 18-50nt.

In another preferred embodiment, the Cas protein is selected from the group consisting of: type II Cas protein, type V Cas protein, type VI Cas protein, or combinations thereof.

In another preferred embodiment, the type II Cas protein is Cas9.

In another preferred embodiment, the Cas protein type II is selected from the group consisting of: a type II-a Cas protein, a type II-B Cas protein, a type II-C variant Cas protein, or a combination thereof.

In another preferred embodiment, the Cas9 is selected from the group consisting of: spCas9, fnCas, saCas9, nmCas9, st1Cas9, st3Cas9, cjCas9, or combinations thereof.

In another preferred embodiment, the Cas9 is derived from one or more selected from the group consisting of: streptococcus pyogenes (SpCas 9), streptococcus thermophilus (Streptococcus thermophiles) (St 1Cas9, st3Cas 9), streptococcus mutans (Streptococcus mutans), staphylococcus aureus (Staphylococcus aureus) (SaCas 9), campylobacter jejuni (Campylobacter jejuni) (CjCas 9), francissamini new jersey (FRANCISELLA NOVICIDA) (FnCas 9), neisseria meningitidis (NEISSERIAMENINGITIDES) (NmCas 9), or a combination thereof.

In another preferred embodiment, the type VI Cas protein is Cas13.

In another preferred embodiment, the Cas protein type VI is selected from the group consisting of: a VI-a type Cas protein, a VI-B type Cas protein, a VI-D type Cas protein, a VI-X type Cas protein, a VI-Y type Cas protein, or a combination thereof.

In another preferred embodiment, the Cas protein type VI is selected from the group consisting of: cas13a, cas13b, cas13c, cas13d, cas13e, cas13f, cas13x, cas13y, or a combination thereof.

In some embodiments, the Cas13 protein is or is derived from the following species: exacter (ALISTIPES), exacter anaerobacter (Anaerosalibacter), bacteroides (bacterioides), bacteroides (Bacteroidetes), burjie (Bergeyella), bluet (Blauthia), vibrio (Butyrivibrio), carbon dioxide fibrinopsis (Capnocytophaga), carnivorous (Carnobacterium), The genus Pseudomonas (Chloroflexus), the genus Flavobacterium (Chryseobacterium), the genus Clostridium (Clostridium), demequina, the family Ubbelobacteriaceae (Eubacteriaceae), the genus Ubbelobacteriaceae (Eubacterium), the genus Flavobacterium (Flavobacterium), the genus Fusobacterium (Fusobacterium), the genus Herbinix, insolitispirillum, the family Ubbelobacteriaceae (Lachnospiraceae), the family Ubbelobacteriaceae (Ubbelobacteriaceae), ciliated (Leptotrichia), listeria (Listeria), aromatic (Myroides), bacillus (Paludibacter), bacterial (Phaeodactylibacter), rhodomonasceae (Porphyromonadaceae), porphyromonas (porphirimonas), prasuvorexa (Prevotella), vibrio pseudobutyrate (Pseudobutyrivibrio), campylobacter (Psychroflexus), Lai Xingshi Bacillus (Reichenbachiella), rhodobacillus (Rhodobacillus), richterus (RIEMERELLA), sinomicrobium, hairyveromyces (Thalassospira), ruminococcus (Ruminococcus); Preferably, ciliates (Leptotrichiashahii), listeria stonecrop (LISTERIA SEELIGERI), mao Luoke bacteria (Lachnospiraceaebacterium) (e.g. Lb MA2020, lb NK4A179, lb NK4A 144), clostridium aminophilum (e.g. CaDSM 10710), chicken bacilli (Carnobacterium gallinarum) (e.g. Cg DSM 4847), and, Propionibacterium (Paludibacter propionicigenes) (e.g., pp WB 4), wei Site Listeria still (LISTERIAWEIHENSTEPHANENSIS) (e.g., lw FSL R9-0317), listeria (Listeriaceaebacterium) (e.g., lb FSL M6-0635), sphingomonas vaccinum (Leptotrichia wadei) (e.g., lw F0279), rhodobacillus capsulatus (Rhodobacter capsulatus) (e.g., rc SB 1003), Rc R121, rc DE 442), oral ciliates (Leptotrichia buccalis) (e.g., lb C-1013-b), herbinix hemicellulosilytica, ubbelobacteriaceae bacteria (Eubacteriaceae bacterium) (e.g., eb CHKCI 004), blautia sp Marseille-P2398, ciliated sp Leptotrichia sp oral classification unit 879str.F0557, Aggregated green-harpoon bacteria (Chloroflexus aggregans), demequina aurantiaca, sea-tangle species (Thalassospirasp.) TSL5-1, vibrio pseudobutyrate (Pseudobutyrivibrio sp.) OR37, vibrio butyricum (Butyrivibrio sp.) YAB3001, ciliated species (Leptotrichia sp.) Marseille-P3007, bacteroides ihuae, and, ZOR0009, bacteroides (Bacteroides pyogenes) (e.g., bp F0041), bacteroides (Bacteroidetes bacterium) (e.g., bb GWA2_31_9), ZOR 00024, LISTERIA RIPARIA, insolitispirillum peregrinum, bacteroides (ALISTIPES sp.), ZOR0009, and ZONE, Animal ulcer Bacillus (Bergeyellazoohelcum) (such as Bz ATCC 43767), canine carbon dioxide biting phage (Capnocytophaga canimorsus), binodimeter carbonic acid phage (Capnocytophaga cynodegmi), chryseobacterium carnipullorum, golden yellow island bacillus (Chryseobacterium jejuense), gill-philic flavobacterium (Chryseobacteriumureilyticum), Flavobacterium bifidus (Flavobacterium branchiophilum), flavobacterium columniform (Flavobacterium columnare), flavobacterium sp 316, myroidesodoratimimus (e.g., Mo CCUG 10230、Mo CCUG 12901、Mo CCUG 3837)、Paludibacterpropionicigenes、Phaeodactylibacter xiamenensis、 Porphyromonas gingivalis (Porphyromonasgingivalis) (e.g., pg F0185), Pg F0568, pg JCVI SC001, pg W4087), rhodomonas potato (Porphyromonas gulae), porphyromonas sp COT-052OH4946, prevotella stupefaciens (Prevotella aurantiaca), prevotella buchneri (Prevotella buccae) (e.g., pb ATCC 33574), prevotella falsenii, prevotella intermedia (Prevotella intermedia) (e.g., pi17, pi, PiZT), prevotella pallens (e.g., pp ATCC 700821), prevotella pleuritidis, prevotella catarrhalis (Prevotella saccharolytica) (e.g., ps F0055), prevotella species (Prevolella sp.) MA2016, prevolella sp.) MSX73, prevolvulella species (Prevolella sp.) P4-76, prevolella sp.) P5-119, Prevolvulella sp.) P5-125, prevolvulella sp.P 5-60, achromobacter torticola (Psychroflexus torquis), reichenbachiellaagariperforans, riemerella anatipestifer (RIEMERELLA ANATIPESTIFER), sinomicrobium oceani, fusobacterium necroseum (Fusobacterium necrophorum) (e.g., fn subsp. Funduliforme ATCC 51357), FnDJ-2, fn BFTR-1, fn subsp.Funduliferam), F.gangrene (Fusobacterium perfoetens) (e.g., fp ATCC 29250), F.ulcerans (Fusobacterium ulcerans) (e.g., fu ATCC 49185), anaerobic salivary species (Anaerosalibacter sp.) ND1, A.nitrite (Eubacterium siraeum), A.xanthus (Ruminococcus flavefaciens) (e.g., rfx XPD 3002), Ruminococcus albus (Ruminococcusalbus) or a combination thereof.

In another preferred embodiment, the Cas13a protein is selected from the following group ：LwaCas13a,LbaCas13a,LshCas13a,PprCas13a,EreCas13a,LneCa13a,CamCas13a,RcaCas13a,HheCas13a,LbuCas13a,LseCas13a,LbmCas13a,LbnCas13a,RcsCas13a,RcrCas13a,RcdCas13a,CgCas13a,Cg2Cas13a,L weCas13a,LbfCas13a,Lba4Cas13a,Lba9Cas13a,LneCas13a,HheCas13a、RcaCas13a or a combination thereof.

In another preferred embodiment, the Cas13a protein is selected from the following species: bacteroides (bacteriodes), bluetermyces (Blueria), vibrio (Butyrivibrio), carnivorous (Carnobacterium), viridifaciens (Chloroflexus), clostridium (Clostridium), demequina, ubbelopsis (Eubacterium), herbinix, insolitispirillum, mahalaridae (Lachnospiraceae), and, Ciliated genus (Leptotrichia), listeria genus (Listeria), bacillus (Paludibacter), rhodomonasceae family (Porphyromonadaceae), vibrio pseudobutyrate genus (Pseudobutyrivibrio), rhodobacter genus (Rhodobacter) or gyrus genus (Thalassospira); Preferably, ciliates (Leptotrichiashahii), listeria stonecrop (LISTERIA SEELIGERI), mao Luoke bacteria (Lachnospiraceaebacterium) (e.g. Lb MA2020, lb NK4A179, lb NK4A 144), clostridium aminophilum (e.g. CaDSM 10710), chicken bacilli (Carnobacterium gallinarum) (e.g. Cg DSM 4847), and, Propionibacterium (Paludibacter propionicigenes) (e.g., pp WB 4), wei Site Listeria still (LISTERIAWEIHENSTEPHANENSIS) (e.g., lw FSL R9-0317), listeria (Listeriaceaebacterium) (e.g., lb FSL M6-0635), sphingomonas vaccinum (Leptotrichia wadei) (e.g., lw F0279), rhodobacillus capsulatus (Rhodobacter capsulatus) (e.g., rc SB 1003), Rc R121, rc DE 442), oral ciliates (Leptotrichia buccalis) (e.g., lb C-1013-b), herbinix hemicellulosilytica, ubbelobacteriaceae bacteria (Eubacteriaceae bacterium) (e.g., eb CHKCI 004), blautia sp Marseille-P2398, ciliated sp Leptotrichia sp oral classification unit 879str.F0557, Aggregated green-harpoon bacteria (Chloroflexus aggregans), demequina aurantiaca, sea-tangle species (Thalassospirasp.) TSL5-1, vibrio pseudobutyrate (Pseudobutyrivibrio sp.) OR37, vibrio butyricum (Butyrivibrio sp.) YAB3001, ciliated species (Leptotrichia sp.) Marseille-P3007, bacteroides ihuae, and, Monomonas bacteria (Porphyromonadaceae bacterium) (e.g., pbKH CP3 RA), LISTERIA RIPARIA, insolitispirillum peregrinum, or combinations thereof.

In another preferred embodiment, the Cas13b is selected from the group consisting of: cas13b-t1, cas13b-t2, cas13b-t3, cas13b-t4, cas13b-t5, cas13b-t6, or combinations thereof.

In another preferred embodiment, the Cas13b is selected from the following group ：BzCas13b,PbCas13b,PspCas13b,RanCas13b,PguCas13b,PsmCas13b,CcaCas13b,AspCas13b,PauCas13b,Pin2Cas13b,、Pin3Cas13b or a combination thereof.

In another preferred embodiment, the Cas13b is or is derived from the following species: exacter (ALISTIPES), bacteroides (Bacteroides), bacteroides (Bacteroidetes), berjie (Bergeyella), carbon dioxide phagostimulant (Capnocytophaga), flavobacterium (Chryseobacterium), flavobacterium (Flavobacterium), flavobacterium (Myroides), bacillus (Paludibacter), bacillus (Bacillus sp), Bacterial bacillus (Phaeodactylibacter), porphyromonas (porphyrimonas), prevotella (Prevotella), clodroxiella (Psychroflexus), lai Xingshi bacillus (Reichenbachiella), rickettsia (RIEMERELLA) or Sinomicrobium; Preferably, the species of genus Eimeria (ALISTIPES sp.) ZOR0009, bacteroides pyogenes (Bacteroides pyogenes) (e.g., bp F0041), bacteroides bacteria (Bacteroidetes bacterium) (e.g., bbGWA2 _31_9), animal ulcer Bacillus (Bergeyella zoohelcum) (e.g., bz ATCC 43767), canine carbon dioxide biting C.sub.2 (Capnocytophaga canimorsus), Xenodime-carbonic acid cytophaga (Capnocytophagacynodegmi), chryseobacterium carnipullorum, golden yellow bacillus of Jizhou island (Chryseobacteriumjejuense), gill-philic flavobacterium (Chryseobacterium ureilyticum), mycobacterium (Flavobacterium branchiophilum), cylindrical flavobacterium (Flavobacterium columnare), Flavobacterium sp 316, myroides odoratimimus (e.g., mo CCUG 10230, mo CCUG12901, mo CCUG 3837), propionibacterium Paludibacter propionicigenes, phaeodactylibacterxiamenensis, porphyromonas gingivalis Porphyromonas gingivalis (e.g., pg F0185), Pg F0568, pgJCVI SC001, pg W4087), rhodomonas potato (Porphyromonas gulae), porphyromonas sp (Porphyromonas sp.) COT-052OH4946, prevotella stutzeri (Prevotella aurantiaca), prevotella buchneri (Prevotella buccae) (e.g., pb ATCC 33574), prevotella falsenii, prevotella intermedia (Prevotella intermedia) (e.g., pi 17, piZT), prevotella intermedia (i.e., pi 3, pi ZT), prevotella buchneri (i.e., prevotella buchneri), Prevotella pallens (e.g., pp ATCC 700821), prevotella pleuritidis, prevotella saccharolytica (Prevotella saccharolytica) (e.g., ps F0055), prevotella sp MA2016, prevotella sp MSX73, prevotella sp P4-76, prevotella sp P5-119, Prevolvulella sp.) P5-125, prevolvulella sp.P 5-60, achromobacter torticola (Psychroflexus torquis), reichenbachiella agariperforans, riemerella anatipestifer (RIEMERELLA ANATIPESTIFER), sinomicrobium oceani, or combinations thereof.

In another preferred embodiment, the Cas13c protein is or is derived from the following species: fusobacterium (Fusobacterium) or anaerobic salivarius (Anaerosalibacter); preferably, the F.necroseum (Fusobacterium necrophorum) (e.g., fn subsp. Funduliforme ATCC 51357, fn DJ-2, fnBFTR-1, fn subsp. Funduliforme), F.gangrene (Fusobacterium perfoetens) (e.g., fp ATCC 29250), F.ulcer (Fusobacterium ulcerans) (e.g., fu ATCC 49185), an anaerobic salivary species (Anaerosalibacter sp.) ND1, or a combination thereof.

In another preferred embodiment, the Cas13d is selected from the group consisting of: rspCas13d, rfxCas d, esCas d, admCas d, or combinations thereof.

In another preferred embodiment, the Cas13d is derived from the following species: eubacterium (Eubacterium) or ruminococcus (Ruminococcus), preferably Eubacterium nitrite (Eubacteriumsiraeum), ruminococcus flavus (Ruminococcus flavefaciens) (e.g., rfx XPD 3002), ruminococcus albus (Ruminococcus albus), or combinations thereof.

In another preferred embodiment, the Cas protein is a V-type Cas protein; or the Cas protein is a Cas protein having a RuvC structure at the C-terminus.

In another preferred embodiment, the V-type Cas protein is Cas12 or Cas protein with RuvC structure at the C-terminus is Cas12.

In another preferred embodiment, the V-type Cas protein is selected from the group consisting of: sup>A V-type Sup>A Cas protein, sup>A V-type B Cas protein, sup>A V-type C Cas protein, sup>A V-type D Cas protein, sup>A V-type E Cas protein, sup>A V-type F Cas protein, sup>A V-type G Cas protein, sup>A V-type H Cas protein, sup>A V-type I Cas protein, sup>A V-type J Cas protein, sup>A V-type K Cas protein, sup>A V-type L Cas protein, or Sup>A combination thereof;

Or the V-type Cas protein is selected from the group consisting of: cas12a, cas12b, cas12c, cas12d, cas12e, cas12f, cas12g, cas12h, cas12i, cas12j, cas12k, cas12l, or a combination thereof;

In another preferred embodiment, the Cas12a is selected from the following group ：FnCas12a、LbCas12a、ErCas12a、Evcas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a、CeCas12a、PrCas12a、CsbCas12a、BhCas12a、SsCas12a、Lb3Cas12a、BpCas12a、PdCas12a、BfCas12a、PcCas12a、cMtCas12a、PeCas12a、LiCas12a、Lb2Cas12a、PmCas12a、MbCas12a、EeCas12a、CsbCas12a、ArCas12a、BsCas12a、AbCas12a、AsCas12a、 or a combination thereof.

In another preferred embodiment, the source of Cas12a is selected from the group consisting of: ciliates, listeria, corynebacteria, sarium, legionella, treponema, actinomycetes, eubacteria, streptococcus, lactobacillus, mycoplasma, bacteroides, flaviivola, flavobacterium, azoospira, sphaerochaeta, gluconacetobacter, neisseria, rochanteria, parvibaculum, staphylococci, nitratifractor, mycoplasma, campylobacter, chaetomium, or combinations thereof.

In another preferred embodiment, the source of Cas12a is selected from the group consisting of: francisella tularensis (FRANCISELLA TULARENSIS) (FnCas a), amino acid coccus BV3L6 (Acidaminococcus sp.BV3L 6) (AsCas a), mahalaceae bacteria ND2006 (Lachnospiraceae bacterium ND 2006) (LbCas a), mahalaceae bacteria NC2008 (Lachnospiraceae bacterium NC 2008) (Lb 5Cas12 a), a method of producing the same, Leuconostoc mesenteroides (Helcococcus sp kunzii) (HkCas a), oribacterium sp.NK2B42 (OsCas a), thiomicrospira sp.XS5 (TsCas a), bacteroides KA00251 (Bacteroidales bacterium KA 00251) (BbCas a), bacteroides stomatitis (Bacteroidetes oral taxon 274) (BoCas a), and, The bacteria MC2017 (Lachnospiraceae bacterium MC 2017) (Lb 4Cas12 a), phlebsiella multocida (Coprococcus eutactus) (CeCas a), propionibacterium rumbet (Prevotella ruminicola strain BPI-34) (PrCas a), candidatus Saccharibacteria bacterium (CsbCas 12 a), vibrio henryi (Butyrivibrio hungatei strain MB 2003) (BhCas a), vibrio parapsilosis (Amersham) and Propionibacterium sp, Smith SCJK08D17 (SMITHELLA sp.SCJK08D17) (SsCas a), trichosporon MC2017 (Lachnospiraceae bacterium MC 2017) (Lb 3Cas12 a), vibrio ruminae (Bytyrivibrio proteoclasticus) (BpCas 12 a), proteus (Prevotella disens) (PdCas 12 a), vibrio fibrinolyticus MD2001 (Butyrivibrio fibrisolvens MD 2001) (BfCas a), vibrio fibrinolyticus, Porphyromonas canis (Porphyromonas crevioricanis) PcCas a, candidatus Methanoplasma termitum (CMtCas a), proteus catarrhalis (Peregrinibacteria bacterium) (PeCas a), leptospira inadaiserovar Lyme (LiCas a), proteus MA2020 (Lachnospiraceae bacterium MA 2020) (Lb 2Cas12 a), proteus pinnatifida, Porphyromonas kii (Porphyromonas macaca) (PmCas a), moraxella bovis (Moraxella bovoculi 237) (MbCas a), eubacterium parvulum (Eubacterium eligens) (EeCas a), candidatus Saccharibacteria bacterium (CsbCas a), eubacterium rectum (Eubacte riumrectale) (ErCas a), eubacterium parvulum (3562 a), rectal yeasts (Agathobacter rectalisstrain) (ArCas a), vibrio butyricum NC3005 (Butyrivibrio sp.nc3005) (BsCas a), toxobacter buchnii (Arcobacter butzleri) (AbCas 12 a) or combinations thereof.

In another preferred embodiment, the source of Cas12b is selected from the group consisting of: alicyclic bacillus calickii (Alicyclobacillus kakegawensis), bacillus V3-13 species, bacillus exovillans (Bacillus hisashii), bacteria of the class globus fulgidus (LENTISPHAERIA BACTERIUM), bacteria of the genus renieratia (LACEYELLA SEDIMINIS), or combinations thereof.

In another preferred embodiment, the Cas12b is selected from the group consisting of: aacCas12b, aaCas12b, bthCas12b, aapCas12b, akCas12b, amCas12b, bs3Cas12b, lsCas12b, or combinations thereof.

In another preferred embodiment, the system I further comprises:

c) A Cas protein trans-cleavage reporter having a single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule that does not hybridize to a guide sequence of a crRNA of the Cas protein.

In another preferred embodiment, the Cas protein trans-cleavage reporter is selected from the group consisting of: a single stranded nucleic acid molecule, a single stranded nucleic acid analog molecule, or a combination thereof.

In another preferred embodiment, the single stranded nucleic acid molecule is selected from the group consisting of: an unmodified single stranded nucleic acid molecule, a modified single stranded nucleic acid molecule, a single stranded nucleic acid molecule containing an abasic spacer, or a combination thereof.

In another preferred embodiment, the Cas protein trans-cleavage reporter is selected from the group consisting of: gold nanoparticle-based reporter molecules, fluorophore-based reporter molecules, fluorescence polarization-based reporter molecules, colloidal phase change/dispersion-based reporter molecules, electrochemical signal-based reporter molecules, semiconductor signal-based reporter molecules, color-based reporter molecules.

In another preferred embodiment, the Cas protein trans-cleaves a reporter, i.e., a nucleic acid probe.

In another preferred embodiment, the nucleic acid probe is provided with a detectable label.

In another preferred embodiment, the fluorescent moiety and the quenching moiety are each independently located at the 5 'end, 3' end, and middle of the nucleic acid of the Cas protein trans-cleaved nucleic acid probe.

In another preferred embodiment, the nucleic acid probe has a length of 3 to 300nt, preferably 5 to 100nt, more preferably 5 to 50nt, most preferably 5 to 15nt.

In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA or single-stranded RNA.

In another preferred embodiment, the nucleic acid probe comprises a single-stranded DNA or single-stranded RNA with a detectable label.

In another preferred embodiment, the single-stranded DNA is fluorescence-and biotin-labeled single-stranded DNA or single-stranded RNA.

In another preferred embodiment, the single-stranded DNA is a fluorescent-labeled single-stranded DNA or single-stranded RNA.

In another preferred embodiment, when the Cas protein is Cas12b or Cas9, the I-th system further comprises a tracrRNA.

In a second aspect, the invention provides a kit for detecting a target nucleic acid molecule, the kit comprising:

a first kit for detecting crRNA prepared from a target nucleic acid molecule, the kit comprising:

i) A first container and a Cas protein located within the first container;

ii) a second container and a Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter within the second container, the Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter comprising a sequence identical or complementary to the target nucleic acid molecule sequence, the Cas protein cis-cleavage substrate not being the target nucleic acid molecule;

iii) Optionally, a third container and a Cas protein trans-cleavage reporter within the third container, the Cas protein trans-cleavage reporter having a single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule that does not hybridize to the guide sequence of the crRNA of the Cas protein.

In another preferred embodiment, the kit further comprises a second kit, the second kit being a crRNA synthesis kit, the kit comprising:

v) a fourth container and an RNA polymerase within the fourth container;

vi) a fifth container and a primer located in the fifth container, the primer structure being: 5'-Z1-Z2-Z3-3';

Wherein, Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

z3' is a sequence capable of hybridizing to a portion of the target nucleic acid molecule or its complement;

vii) a sixth container and a primer within the sixth container, the primer having a structure of 5'-Z4-3', Z4 being a sequence capable of hybridizing to a portion of the target molecule or its complement.

In another preferred embodiment, the second kit further comprises one or more containers selected from the group consisting of:

viii) a seventh container and a reverse transcriptase located within the seventh container;

ix) an eighth container and a DNA polymerase located within the eighth container.

Preferably, the second kit further comprises: x) a ninth vessel and an RNase H enzyme located in the ninth vessel.

In another preferred embodiment, the Cas protein cis-cleavage substrate is selected from the group consisting of: single-stranded DNA, double-stranded DNA, single-stranded RNA, or a combination thereof.

In another preferred embodiment, the Cas protein trans-cleavage reporter has a single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule that does not hybridize to the guide sequence of the crRNA of the Cas protein, selected from the group consisting of: a single stranded nucleic acid molecule, a single stranded nucleic acid analog molecule, or a combination thereof.

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

In another preferred embodiment, any two, three, or four or five or six (or all) of the first, second, third, fourth, fifth and sixth containers may be the same (or the same) or different containers.

In another preferred embodiment, any two, three, or four or five or six or seven or eight or nine (or all) of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth containers may be the same (or the same) or different containers.

In another preferred embodiment, the kit further comprises a buffer.

In a third aspect, the present invention provides a method for detecting a target nucleic acid molecule, comprising the steps of:

(i) Contacting a sample to be detected with a crRNA synthesis system;

(ii) Contacting the product of step (I) with a crRNA detection system comprising a system I comprising:

cas protein; and

A Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter comprising a sequence identical or complementary to a sequence of the target nucleic acid molecule, the Cas protein cis-cleavage substrate not being the target nucleic acid molecule;

(iii) And detecting the activation of the cleavage activity of the Cas protein, so as to judge whether the target nucleic acid molecule exists in the sample.

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule, the crRNA synthesis system comprises a II system, the II system is an RNA amplification system, the II system comprises:

d) A reverse transcriptase;

e) A primer, the primer structure is as follows:

primer 1:5'-Z1-Z2-Z3-3';

primer 2:5-Z4-3;

f) An RNA polymerase;

wherein, Z1 is an RNA polymerase specific recognition sequence;

Z2 is the complete or partial DR sequence;

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule, the crRNA synthesis system comprises a II 'system and a III' system, the crRNA synthesis system is selected from group a 'or group B', the step i) contacting the sample to be detected with the crRNA synthesis system is contacting the sample to be detected with the II 'system first and then contacting the product with the III' system;

wherein group a' is as follows:

d') a reverse transcriptase;

h') a primer, wherein the primer structure is as follows: 5' -Z4' -3';

e') a primer, wherein the primer structure is as follows: 5' -Z1' -Z2' -Z3' -3'; and

F') RNA polymerase;

wherein, Z1' is an RNA polymerase specific recognition sequence;

Z2' is the complete or partial DR sequence;

Wherein group B' is as follows:

d') a reverse transcriptase;

e') a primer, wherein the primer structure is as follows: 5' -Z4' -3';

f ") RNA polymerase;

wherein, Z1' is an RNA polymerase specific recognition sequence;

Z2' is the complete or partial DR sequence;

In another preferred embodiment, the target nucleic acid molecule is a target RNA molecule, the crRNA synthesis system comprises a II "system, a III" system and an IV "system, the crRNA synthesis system is selected from group a" or group B ", the step i) contacting the sample to be detected with the crRNA synthesis system is contacting the sample to be detected with the II" system first, contacting the product with the III "system, and contacting the product with the IV" system;

wherein group a "is as follows:

d') reverse transcriptase;

h ") primer, the primer structure is: 5' -Z4' -3';

The III "system is a cDNA amplification system, the III" system comprising:

e ") a primer, the primer structure being: 5' -Z1' -Z2' -Z3' -3'; and

F ") a DNA polymerase;

wherein, the Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

the IV "system is a DNA transcription system, the IV" system comprising:

g ") RNA polymerase;

preferably, when the reverse transcriptase has no function of digesting RNA, the II "system further comprises an RNase H enzyme; .

Wherein group B "is as follows:

d') reverse transcriptase;

h ") primer, the primer structure is: 5' -Z1' -Z2' -Z3' -3';

wherein, the Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

The III "system is a cDNA amplification system, the III" system comprising:

e ") a primer, the primer structure being:

And

F ") a DNA polymerase;

the IV "system is a DNA transcription system, the IV" system comprising:

g ") RNA polymerase;

In another preferred embodiment, the target nucleic acid molecule is a target DNA molecule, the crRNA synthesis system comprises a III '"system and an IV'" system, and the step i) is to contact the sample to be detected with the crRNA synthesis system by contacting the sample to be detected with the III '"system and then contacting the product with the IV'" system;

The III '"system is a DNA amplification system, the III'" system comprising:

e') primer, the primer structure is as follows:

primer 1:5 '-Z1' -Z2 ',' Z3 '-3',

Primer 2: 5-Z4' -3;

And

F' ") DNA polymerase;

wherein, the Z1' "is an RNA polymerase specific recognition sequence;

Z2' "is the complete or partial DR sequence;

g' ") RNA polymerase.

In another preferred embodiment, the system I further comprises:

A Cas protein trans-cleavage reporter having a single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule that does not hybridize to a guide sequence of a crRNA of the Cas protein.

In another preferred embodiment, the detection of activation of the cleavage activity of the Cas protein is detected by a Cas protein cis cleavage reporter or a Cas protein trans cleavage reporter signal change.

In another preferred embodiment, the activating of the detecting the cleavage activity of the Cas protein is detecting whether the Cas protein cis cleavage substrate or Cas protein cis cleavage reporter is cleaved by Cas protein; or (b)

Detecting whether the Cas protein trans-cleavage reporter is cleaved by a Cas protein.

In another preferred embodiment, cleavage of the Cas protein cis cleavage substrate or the Cas protein cis cleavage reporter by Cas protein indicates the presence of a target nucleic acid molecule in the sample; and the Cas protein cis-cleavage substrate or the Cas protein cis-cleavage reporter is not cleaved by Cas protein, then it indicates that no target nucleic acid molecule is present in the sample.

In another preferred embodiment, cleavage of the Cas protein by the Cas protein in trans indicates the presence of the target nucleic acid molecule in the sample; and the Cas protein trans-cleavage reporter is not cleaved by Cas protein, then it indicates that no target nucleic acid molecule is present in the sample.

In another preferred embodiment, the sequence that hybridizes to the target nucleic acid molecule or a portion of its complement (referred to as Z3, Z3', Z3", Z3'") is no more than 16bp (e.g., 5-30 bp), and Z4, Z4', Z4", or Z4'" is not so limited.

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

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

In another preferred embodiment, the sample is derived from a sample obtained by a culture method selected from the group consisting of: cell culture, bacterial culture, virus culture, fungus culture, microorganism culture, organoid culture, in vivo animal enrichment culture and plant culture.

In another preferred embodiment, the method of nucleic acid amplification is selected from the group consisting of: PCR (Polymerase Chain Reaction Amplification ), LAMP (Loop-mediated isothermal Amplification, loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification ), LCR (LIGASE CHAIN reactions, Ligase chain reaction), bDNA (branched DNA Amplification ), NASBA (Nuclear Acid Sequence-Based Amplification, nucleic acid sequence dependent Amplification), SDA (STRAND DISPLACEMENT Amplification ), TMA (Transcription-Mediated Amplification, transcription-mediated Amplification), RCA (Rolling Circle Amplification ), HDA (Helicase-DEPENDENT AMPLIFICATION, helicase dependent amplification), SPIA (SINGLE PRIMER Isothermal Amplification ), NEAR (Nicking Enzyme Amplification Reaction, nicking enzyme amplification reaction), SMAP2 (Smart Amplification Process version 2, Intelligent amplification method version 2), CPA (Cross Priming Amplification, cross primer amplification), MDA (Multiple Displacement Amplification ), RAM (Ramification), cHDA (circular Helicase-DEPENDENT AMPLIFICATION, helicase dependent circular amplification), SMART (SIGNAL MEDIATED Amplification of RNA Technology, Signal-mediated amplification of RNA), 3SR (Self-Sustained Sequence Replication, autonomous sequence replication system), GEAR (Genome Exponential Amplification Reaction ), IMDA (Isothermal Multiple Displacement Amplification, isothermal multiplex displacement amplification), ERA (Enzymatic Recombinase Amplification, enzymatic recombinant isothermal amplification) or a combination thereof.

In another preferred embodiment, the method is used to detect whether a nucleic acid at a target site is at a SNP, a point mutation, a deletion, and/or an insertion.

In another preferred embodiment, the detection in step (ii) comprises a fluorescent detection method.

In another preferred embodiment, the fluorescence detection method uses an enzyme-labeled instrument or a fluorescence spectrophotometer for detection.

In another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a fourth aspect, the invention provides a use of a type II Cas protein, a type V Cas protein or a type VI Cas protein for determining the presence or absence of a target nucleic acid molecule by detecting crrnas; or a composition, product combination, reagent, kit or system for use in the preparation of the method.

In a fifth aspect, the invention provides a Cas protein cis cleavage substrate comprising a sequence that hybridizes to a target nucleic acid molecule or its complement.

In a sixth aspect, the invention provides a use of a Cas protein cis-cleaving substrate for a method of determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing a composition, product combination, reagent, kit or system under the method, preferably for preparing a composition, product combination, reagent, kit or system for direct detection of a target nucleic acid molecule.

In a seventh aspect, the invention provides a use of a Cas protein trans-cleavage reporter for a method of determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a composition, product combination, reagent, kit or system for use in the preparation of the method.

The eighth aspect of the present invention provides a primer, wherein the primer structure is as follows: 5'-Z1-Z2-Z3-3' or 5'-Z4-3';

wherein, Z1 is an RNA polymerase specific recognition sequence;

Z2 is the complete or partial DR sequence;

Z3 is a sequence capable of hybridizing to a portion of the target nucleic acid molecule or its complement;

z4 is a sequence capable of hybridizing to a portion of the target nucleic acid molecule or its complement.

According to a ninth aspect of the present invention there is provided the use of a primer according to the eighth aspect of the present invention; the use of the primers is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a composition, product combination, reagent, kit or system for use in the preparation of the method.

In a tenth aspect the invention provides the use of a reverse transcriptase, RNA polymerase or DNA polymerase for a method of determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a composition, product combination, reagent, kit or system for use in the preparation of the method.

An eleventh aspect of the invention provides a first composition, product combination, reagent, kit or system comprising:

a) Cas protein; and

B) Cas protein cis cleaves the substrate;

wherein the Cas protein cis-cleavage substrate comprises a sequence that can hybridize to a target nucleic acid molecule or its complement.

In another preferred embodiment, the first composition, product combination, reagent, kit or system comprises a Cas protein cis-cleavage reporter, the Cas protein cis-cleavage substrate being part of the Cas protein cis-cleavage reporter; the Cas protein cis-cleavage substrate is selected from the group consisting of: single-stranded DNA, double-stranded DNA, single-stranded RNA, or a combination thereof.

In another preferred embodiment, the first composition, product combination, reagent, kit or system further comprises c) a Cas protein trans-cleavage reporter.

In a twelfth aspect the invention provides the use of a first composition, product combination, reagent, kit or system for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing a product combination, reagent, kit or system under the method; or the use of the first product combination is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing a reagent, kit or system for preparing the method;

The use of the first reagent is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing a kit or system under the method, preferably for preparing a kit or system for direct detection of a target RNA molecule using a Cas protein; or alternatively

The use of the first kit is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing the system under the process; or alternatively

The use of the first system is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA. In a thirteenth aspect the present invention provides a second composition, product combination, reagent, kit or system comprising:

d) A reverse transcriptase;

e) A primer, the primer structure is as follows:

primer 1:5'-Z1-Z2-Z3-3';

primer 2:5-Z4-3;

f) An RNA polymerase;

wherein, Z1 is an RNA polymerase specific recognition sequence;

Z2 is the complete or partial DR sequence;

Z3 is a sequence which hybridizes to a target RNA molecule or a portion of its complement;

Preferably, the second composition, product combination, reagent, kit or system further comprises an RNase H enzyme when the reverse transcriptase has no function of digesting RNA.

Or the second composition, product combination, reagent, kit or system comprises:

d') reverse transcriptase;

h') a primer, wherein the primer structure is as follows: 5' -Z4 ' -3';

the Z4' "is a sequence capable of hybridizing to a portion of the target RNA molecule;

e ") a primer, the primer structure being: 5' -Z1' -Z2' -Z3' -3'; and

F ") a DNA polymerase;

g ") RNA polymerase;

wherein, the Z1' is an RNA polymerase specific recognition sequence;

z2' is the complete or partial DR sequence;

z3' is a sequence that hybridizes to a target RNA molecule or a portion of its complement;

e') primer, the primer structure is as follows:

primer 1:5' -Z1 ') Z2 ' -Z3 ' -3';

primer 2:5' -Z4 ' -3';

And

F' ") DNA polymerase;

g' ") RNA polymerase;

wherein, the Z1' "is an RNA polymerase specific recognition sequence;

Z2' "is the complete or partial DR sequence;

z3' "is a sequence that hybridizes to a target DNA molecule or a portion of its complement;

z4' "is a sequence which hybridizes to a portion of the target DNA molecule or its complement.

In a fourteenth aspect the invention provides the use of a second composition, product combination, reagent, kit or system for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a combination of products, reagents, kits or systems for use in the preparation of the method; or alternatively

The use of the second product combination is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a reagent, kit or system for use in the preparation of the method; or alternatively

The use of the second reagent is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or a kit or system for use in the preparation of the method; or alternatively

The use of the second kit is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA; or for preparing the system under the process; or alternatively

The use of the second system is a method for determining the presence or absence of a target nucleic acid molecule by detecting crRNA.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1 is a schematic diagram of a specific embodiment.

FIG. 2 is a graph showing the change in fluorescence signal of example 1 and its negative control.

FIG. 3 is a graph showing the change in fluorescence signal of example 2 and its negative control.

FIG. 4 is a graph showing the change in fluorescence signal of example 3 and its negative control.

FIG. 5a is a graph showing the change in fluorescence signal of the experiment using EvCas a in example 4 and its negative control.

FIG. 5b is a graph showing the change in fluorescence signal of the experiment using ErCas a in example 4 and its negative control.

FIG. 6a is a graph showing the change in fluorescence signal of the experiment using LbCas a in example 5 and its negative control.

FIG. 6b is a graph showing the change in fluorescence signal of the experiment using FnCas a in example 5 and its negative control.

FIG. 6c is a graph showing the change in fluorescence signal of the experiment using EvCas a in example 5 and its negative control.

Fig. 7 is a graph of the change in fluorescence signal of the experiments using AapCas b, aacas 12b, respectively, and their negative controls in example 6.

FIG. 8 is a graph showing the change in fluorescence signal of example 7 and its negative control.

FIG. 9 is a graph showing the change in fluorescence signal of example 8 and its negative control.

FIG. 10 is a graph showing the change in fluorescence signal of example 9 and its negative control.

FIG. 11 is a graph showing the change in fluorescence signal of example 10 and its negative control.

Fig. 12 is a gel electrophoresis diagram of example 11 for direct detection of target RNA molecules using the cis-cleavage activity of Cas 9. In the figure 1 is a mark; FIG. 2 shows the system with only double-stranded DNA NT1; FIG. 3 shows the addition of transcriptionally generated crRNA to the system; FIG. 4 shows crRNA for TMA in the system; in FIG. 5, TMA product without RNA template was added to the system (blank).

Fig. 13 is a gel electrophoresis diagram of example 12 for direct detection of target RNA molecules using the cis-cleavage activity of Cas12 a.

Detailed Description

The present inventors have conducted extensive and intensive studies to develop, for the first time, a system for detecting crRNA to determine the presence or absence of a nucleic acid target molecule (the system is to be understood in a broad sense, and may be a composition, a product combination, a reagent, a kit, an instrument or a device containing such a composition, a product combination, a reagent, a kit, a mixture formed when the composition, the product combination, the reagent, the kit are used for detection, or the like), and particularly a detection system for directly detecting a target RNA molecule using Cas9 or Cas12 may be realized. By directly detecting RNA, it is meant that the detection object is RNA, not cDNA transcribed from the target RNA molecule. The inventors have generally conceived that processing a sample to be tested in order to obtain a complete or partial crRNA in the presence of a target nucleic acid molecule contained in the sample to be tested; the treated product is contacted with Cas protein, cas protein cis-cleavage substrate (which is selected when gel electrophoresis display result or trans-cleavage is adopted for signal reporting) or Cas protein cis-cleavage reporter molecule (which is selected when cis-cleavage is adopted for signal reporting), whether the cis-cleavage activity of Cas protein is activated or not is detected, and whether target nucleic acid molecules exist in a sample to be detected or not is judged; or the treated product is contacted with Cas protein, cas protein cis-cleavage substrate and Cas protein trans-cleavage reporter molecule, and whether the trans-cleavage activity of the Cas protein is activated is detected so as to judge whether the target nucleic acid molecule exists in the sample to be detected. A first detection system of the detection system comprises (a) a Cas protein; (b) Cas protein cis-cleaving substrate or Cas protein cis-cleaving reporter (e.g., double-stranded DNA, single-stranded DNA, or single-stranded RNA with or without PAM); the method can further comprise (c) a Cas protein trans-cleavage reporter (or called a nucleic acid probe), and of course, the method can also comprise (c) a Cas protein trans-cleavage reporter, and can also detect whether a Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter is cleaved or not; the second detection system of the system comprises a substance that prepares a crRNA from a target nucleic acid molecule, the targeting sequence of the crRNA being identical or complementary to the target nucleic acid molecule. The Cas protein of this particular embodiment is a type II Cas protein, a type V Cas protein, or a type VI Cas protein; in the case of the type II Cas protein and the type V Cas protein, the detection system breaks through the limitation that classical type II Cas proteins and type V Cas proteins can only detect DNA targets. Briefly, this embodiment can detect a target nucleic acid molecule in the presence of a Cas protein type II, V or VI cis-cleavage substrate or Cas protein cis-cleavage reporter molecule by preparing the target nucleic acid molecule into crrnas in real time (if a tracrRNA/scoutRNA is also required to form the guide RNA, then tracrRNA/scoutRNA is additionally added), and in particular, directly detect the target RNA molecule in the sample with a Cas protein type II, V or VI. On this basis, the present inventors have completed the present application.

Terminology

CRISPR-Cas: a unique bacterial and archaeal derived genomic element that serves as an adaptive immune defense system against invading phage or foreign nucleic acids. The system consists of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas protein, cas for short).

Limit of detection (LOD): the lowest concentration of analyte in the sample can be consistently detected with a typical 95% certainty probability

Cycle threshold (Ct) value: in a PCR reaction, the fluorescent signal exceeds the background level by the number of cycles required. The Ct value is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the greater the amount of target nucleic acid in the sample). Ct value is less than or equal to 36 and is obviously positive. The suspicious, ambiguous/weakly positive results show Ct values between 37 and 40. This suggests that early or late infection, or environmental pollution, may be represented.

The term "Cas protein" refers to a CRISPR-associated protein, which is a related protein in a CRISPR system.

The term "type II Cas protein" refers to the effector protein of the type II family of CRISPR-associated proteins (CRISPR-associated protein), the most notable of which is Cas9.Cas9 is a protein with two nuclease domains (RuvC and HNH), and cis-cleaves substrate DNA mediated by two RNAs to create a double-strand break (DSB) with a blunt end. The two RNA molecules are crRNA and tracrRNA, respectively. These two RNA molecules can be engineered to combine into one guide RNA molecule (gRNA) with both crRNA and tracrrRNA functions.

The term "V-type Cas protein" refers to an effector protein of the V-type family in CRISPR-associated proteins (CRISPR-associated protein) that has diversity at the N-terminus, but retains one unified RuvC-like endonuclease (RuvC) domain at the C-terminus, derived from TnpB protein (Shmakov,S.,Smargon,A.,Scott,D.,Cox,D.,Pyzocha,N.,Yan,W.,et al.(2017).Diversity and evolution of class 2CRISPR-Cas systems.Nat.Rev.Microbiol.15,169–182.doi:10.1038/nrmicro.2016.184). encoded by an autonomous or non-autonomous transposon, further subdivided into a number of subtypes depending on the different CRISPR effector proteins.

The term "type VI Cas protein" refers to effector proteins of the type VI family in CRISPR-associated proteins (CRISPR-associated protein) with RNA-mediated RNA cleavage activity.

The term "Cas12 Sup>A" (formerly "Cpf 1") refers to crRNA-dependent endonucleases, which are enzymes of type V-Sup>A in the CRISPR system classification.

The terms "Cas12B", "C2C1" are used interchangeably to refer to sgRNA-dependent endonucleases, which are enzymes of type V-B in the CRISPR system classification.

The term "PAM" refers to a protospacer-adjacent motif (protospacer-adjacent motif), a short DNA sequence immediately adjacent to a CRISPR effector protein-targeted DNA sequence, necessary for Cas12a or Cas12b to cleave double-stranded DNA, e.g., PAM for Cas12a is TTTV, PAM for aacas 12b is TTN sequence.

The term "target RNA molecule" is the RNA to be detected or a specific portion thereof.

Cas proteins

As used herein, "Cas protein" refers to a CRISPR-associated protein (some documents translate to CRISPR-Cas effect proteins, CRISPR/Cas effect proteins, CRISPR-Cas effectors, CRISPR/Cas effectors) that can be a type II Cas protein, a type V Cas protein, or a type VI Cas protein. Preferably, a V-type Cas protein, which upon binding to a cis-cleaving substrate under guide RNA guidance forms a ternary complex of Cas protein-guide RNA-cis-cleaving substrate, can induce its trans-cleaving activity, i.e. randomly cleaving single-stranded nucleic acids and their equivalents (nucleic acid equivalents such as nucleic acid analogs).

The Cas protein according to this embodiment may be a protein having cis-cleavage activity, or may be a protein having both cis-cleavage activity and trans-cleavage activity.

The Cas protein of this embodiment may be a type II Cas protein; the Cas protein is selected from the group consisting of: a type II-a Cas protein, a type II-B Cas protein, a type II-C variant Cas protein, or a combination thereof.

Cas proteins described in this particular embodiment include Cas9, such as spCas9, fnCas, saCas9, nmCas9, st1Cas9, st3Cas9, cjCas9, or combinations thereof.

The Cas9 is derived from the following species: streptococcus pyogenes (SpCas 9), streptococcus thermophilus (Streptococcus thermophiles) (St 1Cas9, st3Cas 9), streptococcus mutans (Streptococcus mutans), staphylococcus aureus (Staphylococcus aureus) (SaCas 9), campylobacter jejuni (Campylobacter jejuni) (CjCas 9), francissamini new jersey (FRANCISELLA NOVICIDA) (FnCas 9), neisseria meningitidis (NEISSERIAMENINGITIDES) (NmCas 9), or a combination thereof.

The type VI Cas protein is Cas13.

The Cas protein of type VI is selected from the group consisting of: a VI-a type Cas protein, a VI-B type Cas protein, a VI-D type Cas protein, a VI-X type Cas protein, a VI-Y type Cas protein, or a combination thereof.

The Cas protein of type VI is selected from the group consisting of: cas13a, cas13b, cas13c, cas13d, cas13e, cas13f, cas13x, cas13y, or a combination thereof.

In another preferred embodiment, the Cas13a protein is selected from the following group ：LwaCas13a,LbaCas13a,LshCas13a,PprCas13a,EreCas13a,LneCa13a,CamCa s13a,RcaCas13a,HheCas13a,LbuCas13a,LseCas13a,LbmCas13a,LbnCas13a,RcsCa s13a,RcrCas13a,RcdCas13a,CgCas13a,Cg2Cas13a,LweCas13a,LbfCas13a,Lba4Ca s13a,Lba9Cas13a,LneCas13a,HheCas13a,RcaCas13a, or a combination thereof.

In another preferred embodiment, the Cas13a protein is selected from the following species: bacteroides (bacteriodes), bluetermyces (Blueria), vibrio (Butyrivibrio), carnivorous (Carnobacterium), viridifaciens (Chloroflexus), clostridium (Clostridium), demequina, ubbelopsis (Eubacterium), herbinix, insolitispirillum, mahalaridae (Lachnospiraceae), and, Ciliated genus (Leptotrichia), listeria genus (Listeria), bacillus (Paludibacter), rhodomonasceae family (Porphyromonadaceae), vibrio pseudobutyrate genus (Pseudobutyrivibrio), rhodobacter genus (Rhodobacter) or gyrus genus (Thalassospira); Preferably, ciliates (Leptotrichiashahii), listeria stonecrop (LISTERIA SEELIGERI), mao Luoke bacteria (Lachnospiraceaebacterium) (e.g. Lb MA2020, lb NK4A179, lb NK4A 144), clostridium aminophilum (e.g. CaDSM 10710), chicken bacilli (Carnobacterium gallinarum) (e.g. Cg DSM 4847), and, Propionibacterium (Paludibacter propionicigenes) (e.g., pp WB 4), wei Site Listeria still (LISTERIAWEIHENSTEPHANENSIS) (e.g., lw FSL R9-0317), listeria (Listeriaceaebacterium) (e.g., lb FSL M6-0635), sphingomonas vaccinum (Leptotrichia wadei) (e.g., lw F0279), rhodobacillus capsulatus (Rhodobacter capsulatus) (e.g., rc SB 1003), Rc R121, rc DE 442), oral ciliates (Leptotrichia buccalis) (e.g., lb C-1013-b), herbinix hemicellulosilytica, ubbelobacteriaceae bacteria (Eubacteriaceae bacterium) (e.g., eb CHKCI 004), blautia sp Marseille-P2398, ciliated sp Leptotrichia sp oral classification unit 879str.F0557, Aggregated green-harpoon bacteria (Chloroflexus aggregans), demequina aurantiaca, sea-tangle species (Thalassospirasp.) TSL5-1, vibrio pseudobutyrate (Pseudobutyrivibrio sp.) OR37, vibrio butyricum (Butyrivibrio sp.) YAB3001, ciliated species (Leptotrichia sp.) Marseille-P3007, bacteroides ihuae, and, Monomonas bacteria (Porphyromonadaceae bacterium) (e.g., pbKH CP3 RA), LISTERIA RIPARIA, or Insolitispirillum peregrinum.

In another preferred embodiment, the Cas13b is selected from the following group ：BzCas13b,PbCas13b,PspCas13b,RanCas13b,PguCas13b,PsmCas13b,CcaCas13b,AspCas13b,PauCas13b,Pin2Cas13b,Pin3Cas13b or a combination thereof.

The Cas protein of this embodiment may be a V-type Cas protein; the Cas protein is selected from the group consisting of: sup>A V-type Sup>A Cas protein, sup>A V-type B Cas protein, sup>A V-type C Cas protein, sup>A V-type D Cas protein, sup>A V-type E Cas protein, sup>A V-type F Cas protein, sup>A V-type G Cas protein, sup>A V-type H Cas protein, sup>A V-type I Cas protein, sup>A V-type J Cas protein, sup>A V-type K Cas protein, sup>A V-type L Cas protein, or Sup>A combination thereof; the Cas protein of this particular embodiment includes Cas12, e.g., cas12a, cas12b, cas12c, cas12d, cas12e, cas12f, cas12g, cas12h, cas12i, cas12j, cas12k, cas12l, or a combination thereof.

In particular embodiments, cas proteins referred to herein, such as Cas9, cas12, or Cas13, also encompass functional variants of Cas proteins or homologs or orthologs thereof, such as Cas12a mutants disclosed in chinese patent application CN202210508434.7, can be used in this particular embodiment. "functional variant" of a protein as used herein refers to a variant of such a protein that retains, at least in part, the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Functional variants also include fusion products of such proteins with another nucleic acid, protein, polypeptide or peptide that is not normally associated. Functional variants may be naturally occurring or may be artificial. Advantageous embodiments may relate to engineered or non-naturally occurring V-type DNA targeting effector proteins.

In one embodiment, the type II Cas protein, the type V Cas protein, the type VI Cas protein, or an ortholog or homolog thereof may comprise one or more mutations, and thus the nucleic acid molecule encoding the same may have one or more mutations. The mutation may be an artificially introduced mutation and may include, but is not limited to, one or more mutations in the catalytic domain.

In one embodiment, the V-type Cas protein may be from: ciliates, listeria, corynebacteria, sarium, legionella, treponema, actinomycetes, eubacteria, streptococcus, lactobacillus, mycoplasma, bacteroides, flaviivola, flavobacterium, azoospira, sphaerochaeta, gluconacetobacter, neisseria, rochanteria, parvibaculum, staphylococci, nitratifractor, mycoplasma, campylobacter, chaetomium, or combinations thereof.

Table 1V type family effector properties (from: doi: 10.3389/fcell.2020.622103)

^a V represents A, C, and G.

^b R represents A and G ^C B represents C, G and T.

Guide RNA (gRNA)

As used herein, a "guide RNA" is a mature crRNA fused to a tracrRNA as the guide RNA, or a mature crRNA fused to scoutRNA as the guide RNA, or a crRNA alone as the guide RNA.

In general, the guide RNA can comprise, consist essentially of, or consist of, a sequence of identical repeats (DIRECT REPEAT sequences, also known as DR sequences) and a guide sequence (spacer sequences) in the context of endogenous CRISPR systems. Depending on the Cas protein on which the gRNA depends, the gRNA may include crRNA and tracrRNA (e.g., as in the example of Cas12b in this embodiment), crRNA and scoutRNA, or crRNA alone (e.g., as in the example of Cas12a in this embodiment) in different CRISPR systems. The crRNA and tracrRNA may be fused by artificial engineering to form single guide RNA (sgRNA). In certain instances, the guide sequence is a polynucleotide sequence that has sufficient complementarity to the cis-cleaving substrate DNA to hybridize to the cis-cleaving substrate DNA and to direct specific binding of the CRISPR/Cas protein-guide RNA complex to the cis-cleaving substrate DNA, typically having a sequence length of 12-25 nt. The co-repeat sequence can be folded to form a specific structure (e.g., a stem-loop structure) for Cas protein recognition to form a complex. The targeting sequence need not be 100% complementary to cis-cleaving substrate DNA. The targeting sequence is not complementary to the nucleic acid in the trans-cleaving reporter.

In certain embodiments, when optimally aligned, the degree of complementarity (degree of matching) between the targeting sequence and its corresponding cis-cleaving substrate DNA is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.

The term "homology" or "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position between the two sequences. Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such an alignment can be determined by using, for example, amino acid sequence identity by conventional methods, with reference to, for example, the teachings of Smith and Waterman,1981,Adv.Appl.Math.2:482Pearson&Lipman,1988,Pro.Natl.Acad.Sci.USA85:244,Thompson etal.,1994,Nucleic Acids Res22:467380, et al, by computerized operation algorithms (GAP, BESTFIT, FASTA in Wisconsin Genetics software package, and TFASTA, genetics Computer Group). The default parameters may also be used to determine using BLAST algorithms available from the national center for Biotechnology information (NCBI www.ncbi.nlm.nih.gov /).

Cis (cis) cleavage Activity

Cis-cleavage activity refers to the activity of Cas protein binding to the direct repeat sequence of the guide RNA and recognizing and binding to the substrate under the guidance of the guide sequence of the guide RNA, forming a Cas protein-guide RNA-substrate ternary complex with the substrate, and specifically cleaving the substrate. Such substrates, referred to as Cas protein cis-cleavage substrates.

Trans (trans) cleavage Activity

Trans-cleavage activity refers to the non-specific cleavage activity of a Cas protein on a single-stranded nucleic acid molecule (which may also be an equivalent of a nucleic acid molecule, such as a nucleic acid analogue molecule) in addition to specific cleavage of a substrate by binding of the Cas protein to a co-repeat of a guide RNA and recognition and binding of the substrate, which forms a Cas protein-guide RNA-substrate ternary complex with the substrate, guided by the guide sequence of the guide RNA. If a single-stranded nucleic acid molecule is modified to have a signal reporting function, such a single-stranded nucleic acid molecule is referred to as a trans-cleaving reporter in detection.

Nucleic acid analogues

Nucleic acid analogues are a class of derivatives of RNA and DNA, consisting essentially of phosphates, pentoses and bases, and at least one of which is replaced by some other substance, the major nucleic acid analogues being peptide nucleic acids (peptide nucleic acid, PNA), morpholino (MNA), bridge nucleic acids (bridged nucleic acid, BNA) locked nucleic acids (locked nucleic acid, LNA), ethylene glycol nucleic acids (glycol nucleic acid, GNA) and threose nucleic acids (threose nucleic acid, TNA). Some of these nucleic acid analogs can even undergo biological processes such as replication, translation, etc., in vitro (Brudno,Yevgeny;Birnbaum,Michael E;Kleiner,Ralph E;Liu,David R."An in vitro translation,selection and amplification system for peptide nucleic acids".Nature Chemical Biology.6(2):148–155.doi:10.1038/nchembio.280.PMC 2808706.PMID 20081830).

Detection system

The present embodiment provides a detection system for detecting a target nucleic acid molecule, in particular a detection system for detecting crRNA to determine whether the target nucleic acid molecule is present, the detection system comprising a crRNA synthesis system and a crRNA detection system, the crRNA detection system being the I-th system, comprising:

a) Cas protein;

b) A Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter comprising a sequence identical or complementary to the sequence of the target nucleic acid molecule, the Cas protein cis-cleavage substrate not being the target nucleic acid molecule, the crRNA being prepared from a target nucleic acid molecule;

The crRNA synthesis system is a system for preparing complete or partial crrnas from target nucleic acid molecules, the targeting sequences of which are identical or complementary to the target nucleic acid molecules.

In the specific embodiment, the target nucleic acid molecules are processed into the whole or partial crRNA form in real time in detection by a biological technology, and the target nucleic acid molecules are detected in the presence of a Cas enzyme and a Cas protein cis-cleavage substrate, and particularly, the direct detection of the target RNA molecules can be realized by using Cas9 and Cas 12.

The biological techniques used include RNA amplification-related techniques such as TMA, NASBA, SMART, RT-PCR, RT-LAMP, RT-RPA followed by T7 transcription, etc. to produce the desired crRNA.

The technical scheme of the specific embodiment is shown in fig. 1.

Detection method

This embodiment discloses a method for detecting a target nucleic acid molecule, especially a method for directly detecting a target RNA molecule by Cas9 or Cas12, wherein a target nucleic acid molecule such as a target RNA molecule is prepared into a complete or partial crRNA (if the guide RNA consists of crRNA and tracrRNA or scoutRNA, tracrRNA or scoutRNA is provided), and the guide RNA is mixed with a V-type Cas protein, a trans-cleavage reporter molecule (or called a nucleic acid probe), and a cis-cleavage substrate, and then detected (e.g., fluorescence detection). The direct detection of the target RNA molecule in this embodiment means that the target RNA molecule is not required to be made into cDNA molecules for detection.

The specific embodiment provides a detection method for detecting a target nucleic acid molecule, in particular directly detecting a target RNA molecule by using Cas9 and Cas 12. Once the cis-cleaving substrate (single-stranded or double-stranded), guide RNA, and Cas protein form a ternary complex, the complex will non-specifically cleave the trans-cleaving reporter (trans-cleaving reporter, or nucleic acid probe, including single-stranded DNA molecules or single-stranded RNA molecules, depending on the corresponding Cas protein) in the system.

In this embodiment, a pre-formed (e.g., chemically synthesized) cis-cleaving substrate of the Cas protein is mixed as a first system (which also includes tracrRNA or scoutRNA if the guide RNA consists of crRNA and tracrRNA or scoutRNA) to serve as a second system to be tested based on the preparation of the whole or part of crRNA from the target nucleic acid, e.g., the target RNA molecule, and the first and second systems will form a Cas enzyme ternary complex after mixing, activate the cis activity of the Cas protein while activating the trans activity of the Cas protein, cleave the trans-cleaving reporter (or nucleic acid probe), and determine whether the target nucleic acid molecule, e.g., the target RNA molecule, is present by detecting the signal. Experimental results of the specific embodiment show that complete or partial crRNA can be accurately detected by adding the Cas protein and the cis-cleavage substrate into the detection system, and the nucleic acid probe cannot be cleaved by the Cas protein when the complete or partial crRNA does not exist, so that no signal is generated. Thus, this embodiment can determine whether a target nucleic acid molecule, such as a target RNA molecule, is present in the sample by the presence or absence of crRNA.

A representative nucleic acid probe is a single-stranded DNA or single-stranded RNA having a luminescent group and a quencher group attached to both ends, respectively, so that once the probe is cleaved, the luminescent group can emit light.

In this embodiment, it is known whether the system to be detected contains a target nucleic acid molecule, e.g., a target RNA molecule, by detecting fluorescence.

In this particular embodiment, a suitable Cas protein is a V-type Cas protein, preferably Cas12a, cas12b, cas12f, preferably said Cas12a is preferably FnCas12a、LbCas12a、ErCas12a、Evcas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a、CeCas12a、PrCas12a、CsbCas12a、BhCas12a、SsCas12a、Lb3Cas12a、BpCas12a、PdCas12a、BfCas12a、PcCas12a、cMtCas12a、PeCas12a、LiCas12a、Lb2Cas12a、PmCas12a、MbCas12a、EeCas12a、CsbCas12a、ArCas12a、BsCas12a、AbCas12a、AsCas12a、 or a combination thereof.

In this embodiment, the RNA polymerase is T7RNA polymerase. Other useful RNA polymerases are known to those of skill in the art and include specific contacts (Specific contacts between the bacteriophage T3,T7,and SP6RNA polymerasesand their promoters),J.Biol.Chem.266,645-651) and SP6RNA polymerase between T3RNA polymerase (see Jorgensen, E.D., durbin, R.K., risman, S.S., and MCALLISTER, W.T. (1991) phage T3, T7, and SP6RNA polymerase and their promoters (see Melton, D.A., krieg, P.A., rebagliati, M.R., maniatis, T., zinn, K., and Green, M.R. (1984)) and efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids comprising phage SP6 promoters (Efficient in vitrosynthesis of biologically active RNA and RNA hybridization probes fromplasmids containing a bacteriophage SP6promoter),Nucleic Acids Res.12,7035-7056.).

The detection method of this embodiment can detect nucleic acid molecules of different species, such as mammalian, plant, or microbial, viral nucleic acid molecules. The method of this embodiment is particularly suitable for detecting pathogenic microorganisms, genetic mutations or specific target RNAs.

The method of the embodiment can be used for rapidly and directly detecting whether the sample contains the specific RNA sequence. In addition, the sensitivity of the detection method can be greatly improved by combining with amplification technology (such as TMA, NSBA, SMART, RCA, RT-PCR, RT-LAMP, RT-RPA, etc.).

The components used in the various amplification techniques of the present application are, for example:

NTP, buffer, mg ²⁺, etc. required for RNA amplification, and RNase H required for reverse transcriptase without digestion of single stranded RNA functions;

dNTP, buffer solution, mg ²⁺ and the like required during DNA amplification;

these matters are common general knowledge in the art, so the present application is not specifically described.

The main advantages of this embodiment include:

(1) The inventor develops a detection system capable of directly detecting a target RNA molecule based on a type II Cas protein (Cas 9) and a type V Cas protein (Cas 12), wherein the detection system comprises (a) the Cas protein, and the Cas protein is the type II Cas protein or the type V Cas protein; (b) Optionally trans-cleaving the reporter, or nucleic acid probe; (c) Cis-cleaving substrate DNA or Cas protein cis-cleaving reporter molecules with or without PAM; and (d) RNA synthase, the detection system of the present embodiment breaks through the limitations of classical Cas protein detection of DNA targets, and in short, the present embodiment can directly detect target RNA molecules in a sample in the presence of Cas protein, DNA targets by preparing target RNA molecules as crrnas.

(2) According to the embodiment, the crRNA generated in real time is used as a detection target, the Cas protein cis-cleavage substrate is used as a part of the system, so that the whole detection system is free from the limitation of PAM sites, and the crRNA is produced in real time in the detection process, does not need to be synthesized in advance and stored under strict conditions, so that the storage stability and the transportation stability of the whole system are greatly improved, the production cost is reduced to a certain extent, and the crRNA is more beneficial to popularization and use in remote and resource deficient areas.

(3) When the target nucleic acid molecule is a target RNA molecule, the specific embodiment can enrich the target RNA molecule by a certain technical means in the presence of the target RNA molecule, and directly detect the target RNA in the presence of the Cas protein cis-cleavage substrate (i.e., the prefabricated Spacer nucleic acid sequence).

(4) The Cas protein cis-cleavage substrate of the specific embodiment not only can be a DNA double-chain but also can be a DNA single-chain, is flexible in application, improves the stability of a system, and indirectly improves the performance of a reagent.

(5) The specific embodiment breaks through the range of DNA detection of a classical system of Cas12 and Cas9, and can directly detect target RNA molecules through a technical means. This has a major breakthrough in design flexibility, such as not being limited to PAM sites. The Cas enzyme is conventionally used in combination with preformed crrnas, but RNA preservation conditions are more stringent and more susceptible to environmental influences, such as temperature or rnase a enzyme, thereby affecting the stability and performance of the reagent. If improperly stored, reagent failure is highly likely. In the specific embodiment, DNA is used for replacing RNA as a prefabricated component, so that the stability of the reagent is better protected, in addition, the copy number of RNA contained in each sample to be detected in nature is far higher than that of the DNA, the RNA target is easier to detect than the detection with the DNA as the target, the sensitivity of the reagent is relatively improved, and meanwhile, the available tool box is widened for POCT field detection, so that the detection without amplification is possible. In addition, the specific embodiment breaks through the limitation that the Cas12 cannot directly detect the target RNA molecules.

(6) Compared with the detection of RNA by Cas13, the detection method based on Cas9 and Cas12 in the specific embodiment designs cis-cleavage substrates (DNA) according to target RNA molecules, cas13 designs guide RNA according to target RNA molecules, the design of DNA is more convenient and flexible than the design of RNA, and the DNA is easier to preserve and more stable than RNA; the application has higher sensitivity than Cas13 detection RNA.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise indicated, materials and reagents used in the examples of this embodiment are commercially available products.

Example 1

From LbCas a, pre-chemically synthesized DNA duplex NT2 (as cis-cleaving substrate), a nucleic acid probe was mixed as a first system to use crRNA made in real time by T7 transcriptase as a second system to be tested, and after mixing the first and second systems, a Cas enzyme ternary complex would be formed, activating the cis activity of LbCas a while activating the trans activity of LbCas a, cleaving the nucleic acid probe to reveal the presence or absence of the target RNA molecule.

Materials:

The first system: lbCas12a (10 μm): 1 μl, NT2 (200 nM): 2 μL, NEBuffer ^TM 2.1.1 (from NEW ENGLAND Biolabs): 2. Mu.L, nucleic acid probe (200 nM): 1 mul.

T7 transcription system: t7 promoter (SEQ ID NO: TAATACGACTCACTATAGG, same as in the following examples): 4. Mu.M (final concentration, same as in the examples below), 10 Xbuffer (400 mM Tris-HCl < pH7.9>;100mM DTT < dithiothreitol >;20mM spermidine < SPERMIDINE >;60mM MgCl ₂), 50U/. Mu.l of T7 enzyme (in an amount of 4U/reaction, same as in the examples below).

The nucleic acids used are shown in Table 2.

TABLE 2 nucleic acids used in example 1

The procedure is as follows:

T7-CovidN1 and NTC (No Template Control, namely a negative control experiment, wherein the negative control experiment is an experiment without adding T7-CovidN 1) are reacted for 60Min at 37 ℃ by using a T7 transcription system, 2 mu L of a product is taken as a second system to be added into a first system, fluorescence of FAM channels is collected at 37 ℃ for 1 Min/time in a fluorescence quantitative PCR instrument ABI Qs3, and the circulation is carried out for 30Min.

Results:

As shown in FIG. 2, by adding LbCas a protease, cis-cleaving substrate NT2, and nucleic acid probe to the reaction system, the crRNA produced in real time (see T7 transcription curve in FIG. 2) can be accurately detected, and when no transcribed crRNA is present (i.e., negative control experiment), the nucleic acid probe will not be cleaved by Cas12a enzyme, and no signal will be generated (see NTC curve in FIG. 2).

Example 2

By using LbCas a and synthesized DNA double-stranded NT2 (serving as a cis-cleavage substrate) as a first system, enriching by Rt-PCR, taking crRNA manufactured by T7 transcriptase as a second system to be detected, mixing the two systems to form a ternary complex, starting cis activity of LbCas a, activating trans activity of LbCas a, and cutting a nucleic acid probe to show whether target RNA molecules exist or not.

Materials:

The first system: lbCas12a (10 μm): 1 μl, NT2 (200 nM): 2 μL, NEBuffer ^TM 2.1.1: 2. Mu.L of nucleic acid probe (200 nM) (same as in example 1): 1 mu L

RT-PCR system: takaRa one-step RT-PCR kit

T7 transcription system: t7 promoter, 10 Xbuffer (400 mM Tris-HCl < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂), T7 enzyme 50U/. Mu.l

The nucleic acids used are shown in Table 3.

TABLE 3 nucleic acids used in example 2

The procedure is as follows:

CovidN (RNA) and NTC (i.e. negative control experiment, without adding T7-CovidN 1) were treated according to instructions with Takara one-step RT-PCR kit, primers were subjected to conventional RT-PCR reaction with RT0001 (1 μL) and RT0002 (1 μL), then 2 μL of the product was taken to react at 37℃for 60Min in T7 transcription system, 2 μL of the product was taken as the second system to be added into the first system, FAM channel fluorescence was collected at 37℃for 1 Min/time at ABI Qs3, and the cycle was continued for 30Min.

Results:

Referring to FIG. 3, by adding LbCas a protease, cis-cleaving substrate NT2, and nucleic acid probe to the reaction system, RT-PCR-produced crRNA can be detected, and in the absence of produced crRNA (i.e., negative control experiment), nucleic acid probe will not cleave, and no signal will be generated. Unlike classical Cas12a detection of cis-cleaved substrates as target nucleic acids, we target crrnas generated in real time in the reaction as targets to be detected, thereby detecting the presence or absence of target RNA molecules.

Referring to FIG. 3, this illustration shows that crRNA prepared by RT-PCR and transcriptase works well in the phylogenetic system.

Example 3

The first system is LbCas a, a synthesized DNA double strand (used as a cis-cleavage substrate), the second system is crRNA manufactured by NASBA/TMA, and the two systems are mixed to form a ternary complex, the cis activity of LbCas a is started, the trans activity of LbCas a is activated, and a nucleic acid probe is cleaved, so that whether the target RNA molecule exists or not is displayed.

Materials:

The first system: NT2 (200 nM): 2. Mu.L; lbCas12a (10 μm): 1 μl; NEBuffer ^TM 2.1.1: 2. Mu.L; nucleic acid probe (200 nM): 1 μl; rnase-free water: 14. Mu.L.

TMA buffer (3 x buffer): 120mM Tris-HCl (pH 8.5), 210mM KCl,36mM MgCl ₂, 30mM DTT,45% (V/V, below) DMSO (dimethyl sulfoxide).

10 Xenzyme premix: MMLV reverse transcriptase (from NEW ENGLAND Biolabs, examples below, supra), megadimension T7RNA polymerase (from Shanghai megadimension technology development Co., ltd., examples below)

Transcription system: t7 promoter 10 Xbuffer (400 mM Tris < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂), T7 enzyme 50U/. Mu.l

The nucleic acids used are shown in Table 4.

TABLE 4 nucleic acids used in example 3

The procedure is as follows:

TMA-n0003, TMA-n0004 each 1 μl, TMA buffer, 10 Xenzyme premix, and target RNA molecular template CovidN were configured such that TMA reaction was performed at 42deg.C for 90min, 2 μl of the product was added to the first system, and fluorescence was collected from FAM channels at 37deg.C for 1 min/time at ABI Qs3, and circulated for 30min.

Results:

Referring to FIG. 4, this illustration shows that by adding LbCas a, cis-cleaving substrate NT2, nucleic acid probe, NASBA/TMA method-produced crRNA can be accurately detected, whereas in the absence of produced crRNA (i.e., negative control experiments, which are experiments without the addition of Covidn (RNA)), no cleavage of the nucleic acid probe occurs. Unlike classical Cas12a detection, we use NASBA/TMA processed crRNA as the target to be detected, thereby detecting the presence or absence of target RNA molecules.

The system is as follows: in the presence of the target RNA molecule, the crRNA produced by the system via TMA/NASBA works well, whereas no sample RNA is present and no signal is present throughout the system.

Example 4

The first system is ErCas a/EvCas a, the second system is a crRNA (crRNA) manufactured by T7/TMA (T7/TMA) and used as a second system to be detected, a ternary complex is formed after the two systems are mixed, cis activity of ErCas a/EvCas a is started, trans activity of ErCas a/EvCas12a is activated, and a nucleic acid probe is cut to show whether target RNA molecules exist or not.

Materials and procedures: other materials and procedures were the same as in examples 1 and 3 except for the enzyme.

Results:

Referring to FIGS. 5a and 5b, this illustration shows that TMA-produced crRNA can be accurately detected by adding Ercas a/Evcas12a, cis-cleaving substrate, nucleic acid probe, without cleavage of the nucleic acid probe in the absence of the produced crRNA (negative control). Unlike classical Cas12a detection, we use crRNA generated by NASBA reaction as the target to be detected, thereby detecting the target RNA molecule.

The above example of fig. 5a, 5b illustrates that the present system has versatility in Cas12a proteins.

Examples 1-4 summary

Examples 1-4 illustrate that the methods of direct detection of target RNA molecules of this embodiment have versatility in different Cas12a enzyme systems, whether LbCas a, fnCas a, evacas 12a, erCas12a. These examples illustrate that the design of the present system in terms of detection is relatively flexible compared to classical systems, since the present system does not rely on the original sequence PAM, the design area is wider and the design is more convenient.

Example 5

Materials and procedures: lbCas12A experiment the rest of the materials and procedure were exactly the same as in example 1 above, except that ssDNA was used as cis-cleavage substrate for the reaction material. EvCas12A experiment the rest of the materials and procedure were exactly the same as in example 4 above, except that ssDNA was used as cis-cleavage substrate for the reaction material. FnCas12A experiment, the rest of the materials and procedure were exactly the same as in example 1 above, except that FnCas a was used as the reaction material and ssDNA was used as the cis-cleavage substrate.

The nucleic acids used as cis-cleavage substrates are shown in Table 5.

TABLE 5 nucleic acids used as cis-cleavage substrates for example 5

Results:

Referring to FIGS. 6a, 6b, 6c, ssDNA is also suitable for this embodiment. In combination with examples 1-4, it was demonstrated that Cas12a activity could be well initiated whether single-stranded DNA was synthesized or double-stranded DNA was used as the cis-cleaving nucleic acid target.

There are various ways of preparing crrnas, including but not limited to the ways described in the examples above, such as T ^M a, NASBA, SMART, RCA, etc., which can be used in this embodiment.

Example 6

The Cas12b, the tracrRNA and the synthesized DNA single strand (serving as cis-cleavage substrates) serve as a first system, the crRNA manufactured through transcription serves as a second system to be detected, a ternary complex is formed after the crRNA and the second system are mixed, the cis activity of the Cas12b is started, the trans activity of the Cas12b is activated, a nucleic acid probe is cut, and fluorescence is emitted to detect whether a target RNA molecule exists.

Materials:

The first system: aac/AapCas b (10. Mu.M): 1 μl, cas12b tracrRNA (200 nM): 2 μl, NT4 (200 nM): 2 μL, NEBuffer ^TM 2.1.1: 2. Mu.L, nucleic acid probe (200 nM): 2 mu L

T7 transcription system: t7 promoter, 10 Xbuffer (400 mM Tris < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂), T7 enzyme 50U/. Mu.l

The nucleic acids used are shown in Table 6.

TABLE 6 nucleic acids used in example 6

The procedure is as follows:

T7-CovidN4 and NTC (negative control experiment, negative control experiment is not added with T7-CovidN) are reacted for 60Min at 37 ℃ by using a T7 transcription system, 2 mu L of the product is taken as a second system to be added into a first system, FAM channel fluorescence is collected at the temperature of 37 ℃ for 1 Min/time at ABI Qs3, and the cycle is 30Min.

Results:

Referring to FIG. 7, this illustration shows that the first system can accurately detect the manufactured crRNA without cleavage of the nucleic acid probe in the presence of the manufactured crRNA. Unlike classical Cas12b detection, we use crRNA generated in real time by molecular biology techniques of the target RNA molecule as the target to be detected, thereby detecting the target RNA molecule.

Referring to FIG. 7, this figure and this example illustrate that the detection methods and systems of this embodiment are also applicable to AapCas b and AacCas b enzymes.

Example 7

The first system is AapCas b, cas12btracrRNA, synthetic DNA single chain and nucleic acid probe, the second system is crRNA made by NASBA/TMA, and the two systems are mixed to form a ternary complex, the cis activity of AapCas b is started, the trans activity is activated, and the nucleic acid probe is cut to indirectly detect whether target RNA molecules exist.

Materials:

TMA buffer (3 x buffer): 120mM Tris-HCl (pH 8.5), 210mM KCl,36mM MgCl ₂, 30mM DTT,45% DMSO.

10 Xenzyme premix: MMLV reverse transcriptase, megadimension T7RNA polymerase

Cas12b first system: NT2 (200 nM): 2. Mu.L; cas12 enzyme (10 μm): 1 μl; tracrRNA (200 nM): 1 μl; NEBuffer ^TM 2.1.1: 2. Mu.L; nucleic acid probe (200 nM): 1 μl; rnase-free water: 14. Mu.L.

The nucleic acids used are shown in Table 7.

TABLE 7 nucleic acids used in example 7

The procedure is as follows:

TMA-n0005, TMA-n0006 each 1 μl, TMA buffer, 10 Xenzyme premix, and RNA template CovidN were prepared to allow TMA reaction at 42deg.C for 90min, adding 2 μl of the product into Cas12a first system, setting at 37deg.C for 1min at ABI Qs3, collecting FAM channel fluorescence each time, and circulating for 30min.

Results:

referring to fig. 8, this system illustrates: in the presence of the target RNA molecule, the crRNA produced by the system via TMA/NASBA works well, whereas no sample RNA is present and no signal is present throughout the system.

Example 8

Cas12f (called Cas14a.1 and Cas14 a), the application supports the Cas protein to be incorporated into Cas 12) (PDB ID:7C 7L), tracrRNA, synthesized DNA single-chain is used as a first system, crRNA manufactured through transcription is used as a second system to be tested, a ternary complex is formed after the crRNA and the crRNA are mixed, cis activity of Cas12f is started, trans activity of Cas12f is activated, a nucleic acid probe is cut, and fluorescence is emitted, so that whether target RNA molecules exist or not is detected. Unlike classical Cas12f detection, we use crRNA generated in real-time in response as the target to be detected, thereby detecting the target RNA molecule.

Materials:

the first system: cas12f (10 μm): 1 μl, cas12F TRACRRNA (200 nM): 2 μl, NT5 (200 nM): 2. Mu.L, NEBuffer ^TM 2.1.1, nucleic acid probe (200 nM): 2 mu L

T7 transcription system: t7 promoter, 10 Xbuffer (400 mM Tris < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂); t7 enzyme 50U/. Mu.l

The nucleic acids used are shown in Table 8.

TABLE 8 nucleic acids used in example 8

The procedure is as follows:

T7-CovidN and NTC (negative control without adding T7-CovidN 5) were reacted with T7 transcription system at 37deg.C for 60Min, 2 μL of the product was added as second system into the first system, and FAM channel fluorescence was collected at 37deg.C for 1 Min/time at ABI Qs3, and circulated for 30Min.

Results:

Referring to fig. 9, this illustration shows that this embodiment applies to Cas12f enzymes.

Example 9 preparation of partial crRNA for detection

By taking ErCas a, half-section RNA and synthesized DNA single chain as a first system, taking truncated crRNA manufactured through transcription as a second system to be detected, mixing the two systems to form a ternary complex, starting the cis activity of AacCas b, activating the trans activity of Cas12b, cutting a nucleic acid probe, and emitting fluorescence to determine whether target RNA molecules exist. Unlike classical Cas12b detection, we use the partial crRNA generated in real time in response as the target to be detected, thereby detecting the target RNA molecule.

Materials:

TMA buffer (3 x buffer): 120mM Tris-HCl, pH 8.5, 210mM KCl,36mM MgCl ₂, 30mM DTT,45% DMSO.

10 Xenzyme premix: MMLV reverse transcriptase, megavitamin T7 RNase

Transcription system: a T7 promoter; 10 Xbuffer (400 mM Tris-HCl < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂); t7 enzyme 50U/. Mu.l

The first system: half RNA (UAAUUUCUACU) (200 nM): 1 μl; NT6 (200 nM): 2. Mu.L; erCas12A enzyme (10. Mu.M) 1. Mu.L, NEBuffer ^TM 2.1.1:2. Mu.L, nucleic acid probe (200 nM): 1 μl of rnase-free water: 13 mu L.

The nucleic acids used are shown in Table 9.

TABLE 9 nucleic acids used in example 9

The procedure is as follows:

TMA-n0007, TMA-n0008 each 1 μl, TMA buffer,10 Xenzyme premix, template CovidN of target RNA molecule was configured to react at 42deg.C for 90min, 2 μl of the product was taken as TMA group and added into Cas12a first system, and FAM channel fluorescence was collected at 37deg.C for 1 min/time at ABI Qs3, and circulated for 30min.

T7-CovidN and NTC (no CovidN is added in negative control) are reacted at 37 ℃ for 60Min by using a T7 transcription system, 2 mu L of the product is taken as a transcriptome and NTC group is added into a first system, FAM channel fluorescence is collected at 37 ℃ for 1 Min/time at ABI Qs3, and the cycle is 30Min.

Results:

Referring to FIG. 10, this illustration shows that the crRNA produced may be a truncated crRNA, and that the particular pattern of truncation may be flexible, with the truncated crRNA being preferably a DR loop region, as a portion of the sequence of the complete crRNA is split in half.

Example 10 demonstrates crRNA aptamer system made by ligase transcription

The first system is LbCas a and the synthesized DNA single chain is taken as a first system, the crRNA manufactured by a ligase method is taken as a second system to be detected, the two systems are mixed to form a ternary complex, the cis activity of LbCas a is started, the trans activity of Cas12a is activated, and a nucleic acid probe is cut to detect whether a target RNA molecule exists.

Materials:

ligase system: t4 ligase kit (available from Shanghai megaView technology development Co., ltd.).

Transcription system: t7 promoter, 10 Xbuffer (400 mM Tris-HCl < pH7.9>,100mM DTT,20mM spermidine, 60mM MgCl ₂); t7 enzyme 50U/. Mu.l

The first system: NT2 (200 nM): 2 μl, lbCas a (10 μΜ): 1 μl; NEBuffer ^TM 2.1.1: 2. Mu.L; nucleic acid probe (200 nM): 1 μl; rnase-free water: 14. Mu.L.

The nucleic acids used are shown in Table 10.

TABLE 10 nucleic acids used in example 10

The procedure is as follows:

ligation0010, ligation0011 each 1. Mu.L, ligase buffer, T4 ligase, target RNA molecular template CovidN were configured to ligate the reaction solution to react at 37℃for 90min, 2. Mu.L of the product was added to the transcription system, transcribed at 37℃for 90min, 5ul of the product was taken to the Cas12a primary system, and FAM channel fluorescence was collected at 37℃for 1 min/time at ABI Qs3 and cycled for 30min.

Results:

Referring to FIG. 11, this figure illustrates that crRNA produced by ligase is suitable for use in the system.

Example 11 direct detection of target RNA molecules using cis-cleavage activity of Cas9

Materials:

10 Xenzyme premix: MMLV reverse transcriptase, megadimension T7RNA polymerase

Transcription system: t7 promoter, 10 Xbuffer (400mM Tris pH7.9, 100mM DTT,20mM spermidine, 60mM MgCl ₂), T7 enzyme 50U/. Mu.l

Cas9 cleavage system: cas9 tracrRNA:1 μl, NT1: 2. Mu.L of Cas9 enzyme (10. Mu.M): 1. Mu.L of NEBuffer ^TM 2.1.1:2. Mu.L of nucleic acid probe: 1 μl of rnase-free water: 12. Mu.L.

The procedure is as follows:

A2 nd lane Cas9 cleavage system;

Lane 3: firstly, after T7-CovidN is treated by a transcription system, 2 mu L of a product is taken and added into a Cas9 cutting system;

Lane 4: TMA0001, TMA0002 each 1 μl, TMA buffer,10 Xenzyme premix, and RNA template CovidN to be tested configured into TMA reaction solution, reacting at 42deg.C for 90min, adding 2 μl of the product into Cas9 cleavage system;

Lane 5: TMA buffer,10 Xenzyme premixing, water reaction at 42 ℃ for 90min, adding 2 mu L of product into Cas9 cutting system;

cleavage reaction before electrophoresis: mixing the system to be tested and the prefabricated Cas9 cutting system for 60min at 37 ℃.

Electrophoresis: electrophoresis was performed at 120V for 30min.

Expression 11 list of nucleic acids used in example 11

FIG. 12 shows the CAS9 protein in the present system: the Cas9 enzyme, the double-stranded DNA target as a pre-mixed system and the crRNA prepared from the sample RNA as a system to be tested works well. The Taget channel without crRNA is used as a negative control, crRNA generated by direct transcription is used as a positive control, and crRNA manufactured by TMA through RNA samples is used as a sample to be detected. It should be emphasized that the difference between the present invention and the common Cas9 reaction is that the classical Ca9 pathway is that the sample is a system to be tested using DNA double strand, and the sample is detected with Cas9 enzyme and a pre-prepared crRNA mixed system.

Example 12 direct detection of target RNA molecules using cis-cleavage activity of Cas12a

The Lb/FnCas a is used as a first system, the crRNA manufactured by transcription and NASBA/TMA is used as a second system to be detected, a ternary complex is formed after the crRNA and the crRNA are mixed, the cis activity of the Cas12a is started, the DNA double-strand target of the first system is cut, and whether the target RNA molecule exists or not is judged by detecting whether the DNA double-strand is cut or not. This example uses the cis activity of the Cas12a system to directly detect the target RNA molecule.

Materials:

10 Xenzyme premix: MMLV reverse transcriptase, megadimension T7RNA polymerase

Transcription system: t7 promoter, 10 Xbuffer (400mM Tris PH7.9, 100mM DTT,20mM spermidine, 60mM MgCl2), T7 enzyme 50U/. Mu.l

Cas12a first system: NT10, 2. Mu.L, cas12 enzyme (10. Mu.M): 1. Mu.L, NEBuffer ^TM 2.1.1:2. Mu.L, RNase-free water: 14. Mu.L.

The procedure is as follows: TMA-0003, TMA-0004 each 1 μl, TMA buffer, 10 Xenzyme premix, and RNA template CovidN to be tested were prepared to be TMA reaction solution, reacted at 42deg.C for 90min, adding 2 μl of the product into Lb/FnCas a first system, and electrophoresis was performed in water bath at 37deg.C for 60min, and taking 10 μl of the product. Wherein LbCas a is placed in lane 3 and FnCas a is placed in lane 5.

T7-CovidN1 and NTC were reacted with T7 transcription system at 37deg.C for 60Min, 2 μl of the product was taken as transcriptome and NTC set was added to the first system, and electrophoresis was performed in water bath at 37deg.C for 60Min, 10 μl of the product was taken. Wherein LbCas a is placed in the second lane and FnCas a is placed in lane 4. The first lane was placed with control NT10 and the remaining lanes were placed with DNA markers (mark)

TABLE 12 list of nucleic acids used in example 12

Fig. 13 illustrates cis activity compatible with LbCas a, fnCas a of this embodiment, as another example of application of the invention.

Protein sequences used in this embodiment:

FnCas12a：MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

LbCas12a：MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIA KAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH

ErCas12a：NGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDDYYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETYIHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNNAIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGNIQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYFLHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTDINWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNKRYLKRPAATKKAGQAKKKK

EvCas12a：

MESNNKIFTETIGTSSIAKTMRNSLVPTESTKRNIEKNGIIIDDQLRAEKRQQLKEI

MDEYYRAYIDSKLSNVALTRTIDWKELFQAIENNYKQNTTKTKNELEKKQKEK

RTEIYKILSDDEEFKQLFNAKLLTNILPEFIKNQNIDNEEKQEKISTVELFQRFTSS

FTDFFKNRKNVFSKDEISTSICYRVVQENAWIFYQNLLAFEEIKKTAEQEIKKIEA

ENRDSISDYSLKEIFDFDFYGLLLNQGGIRFYNDVCGKINYHMNLYGQKHNIKS

NKFKMKRMHKQILSIDESTFEVPTMFENDKEVYQVLNEFLSDLASKKILERVEKI

GENVSEYEINKIYIQSKNFENFSSFMCGNWQIINDSLKTYYNEKIKSKGKAKVEK

VKKAIKAIEYKSLADINQLVERYNHDELNRKAEEYISAINEKIKDLYVNEIEFDE

KTNLIENETKSEEIKSKLDSIMEIMHWTKMFIIEEEIEKDVNFYNEIEEIYDELQPL

VTIYNRIRNYVTQKPYSEEKIKLNFGIPTLANGWSKTKEYDNNAIIMIRDGKYYL

GIFNAKNKPDKKIMEGHQSEENGDYKKMIYRLLPGPNKMLPKVFMSKTGIAEY

KPSQYILECYEQNKHIKSDKNFDIKFCRDLIDFFKTSINRHPEWSKFNFKFSETSE

YEDISTFYREVEKQGYKIEWTYISEKEIKELDENGQLYLFQIYNKDFSEKSKGKE

NLHTMYLKNLFSEENLKNIVLKLNGEAEVFFRKSSIKKPIIHKKGSVLVNKTYNE

NGERKSIPEEQYTEIYKYLNSIGTNELSEKSKKLMEEGKVEYYKANYDIVKDYR

YSVDKFFIHLPMTINFKAAGFSPINNIALKSIALKEDMHIIGIDRGERNLIYVSVID

TKGNIVEQRNFNIVNGIDYKEKLKQKELDRDNARKNWKEIGKIKDLKEGYLSL

VVHEIAKLVVKYNAIITMEDLNQGFKRGRFKVERQVYQKFETMLINKLNYLVD

KDLAVDQEGGLLRGYQLTYIPESLKVLGRQCGYIFYVPAAYTSKIDPTTGFVAIF

NYKGMTDKDFVTSFDSIKYDDERGLFAFEFDYENFVTHKVEMARNKWTVYTY

GERIKRKFKNGLWDTAEKVDLTYQMRSILEKYEIEYNKGQDILEQIEELDEKAQ

NGICKEIKYLVKDIVQMRNSLPDNAVEDYDAIISPVINNNGEFFDSTRGDEDKPL

DADANGAYCIALKGLYEVMQIKKNWNEETEFPRKELKIRHQDWFDFIQNKRYLAacCas 12b：GIHGVPAAAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQD SACENTGDIKRPAATKKAGQAKKKK

AapCas12b

GSMAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A target nucleic acid molecule detection system, characterized in that it includes a first system, wherein the first system is a crRNA detection system, and the first system includes:

a) Cas protein;

b) Cas protein cis-cleavage substrate or Cas protein cis-cleavage reporter molecule;

Wherein, the Cas protein cis-cleaves the substrate or the Cas protein cis-cleaves the reporter molecule (including a sequence identical to or complementary to the target nucleic acid molecule sequence;

The Cas protein cis-cleavage substrate is not the target nucleic acid molecule, and the crRNA is prepared based on the target nucleic acid molecule.

2. The target nucleic acid molecule detection system according to claim 1, characterized in that the target nucleic acid molecule is a target RNA molecule, and the Cas protein cis-cleavage substrate includes a sequence that is identical or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system also includes a second system; the second system is a crRNA synthesis system;

The second system is an RNA amplification system, and the second system comprises:

d) reverse transcriptase;

e) a primer, wherein the primer structure is:

Primer 1: 5′-Z1-Z2-Z3-3′;

Primer 2: 5-Z4-3;

f) RNA polymerase;

Wherein, the Z1 is a specific recognition sequence for RNA polymerase;

Z2 is a complete or partial DR sequence;

Z3 is a sequence that can partially hybridize with the target RNA molecule or its complementary sequence;

Z4 is a sequence that can partially hybridize with the target RNA molecule or its complementary sequence;

Preferably, when the reverse transcriptase does not have the function of digesting RNA, the system II further comprises RNase H enzyme.

3. The target nucleic acid molecule detection system according to claim 1, characterized in that the target nucleic acid molecule is a target RNA molecule, and the Cas protein cis-cleavage substrate includes a sequence identical to or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system also includes a II' system and a III' system; the II' system and the III' system constitute a crRNA synthesis system, and the crRNA synthesis system is selected from Group A' or Group B';

Group A’ is as follows:

The II' system is an RNA reverse transcription system, and the II' system comprises:

d’) reverse transcriptase;

h') primer, the primer structure is: 5'-Z4'-3';

The Z4' is a sequence that can partially hybridize with the target RNA molecule;

The III' system is a cDNA transcription and amplification system, and the III' system comprises:

e') a primer, wherein the primer structure is:

5’-Z1’-Z2’-Z3’-3’; and

f’) RNA polymerase;

Wherein, the Z1' is a specific recognition sequence for RNA polymerase;

Z2' is a complete or partial DR sequence;

Z3' is a sequence that can partially hybridize with the target RNA molecule or its complementary sequence;

Preferably, when the reverse transcriptase does not have the function of digesting RNA, the II' system further comprises RNase H enzyme;

Group B’ is as follows:

Wherein the II' system is an RNA reverse transcription system, and the II' system comprises:

d’) reverse transcriptase;

h') a primer, wherein the primer structure is: 5'-Z1'-Z2'-Z3'-3';

e') a primer, wherein the primer structure is: 5'-Z4'-3';

f”) RNA polymerase;

Wherein, the Z1' is a specific recognition sequence for RNA polymerase;

Z2' is a complete or partial DR sequence;

Z3' is a sequence that can hybridize with a portion of the target RNA molecule;

Z4' is a sequence that can partially hybridize with the target RNA molecule or its complementary sequence;

Preferably, when the reverse transcriptase does not have the function of digesting RNA, the II' system also includes RNase H enzyme.

4. The target nucleic acid molecule detection system according to claim 1, characterized in that the target nucleic acid molecule is a target RNA molecule, and the Cas protein cis-cleavage substrate includes a sequence identical to or complementary to the target RNA molecule sequence; the target nucleic acid molecule detection system also includes a "II" system, a "III" system, and a "IV" system; the "II" system, the "III" system, and the "IV" system constitute a crRNA synthesis system, selected from the "A" group or the "B" group;

Group A:

The "II" system is an RNA reverse transcription system, and the "II" system includes:

d”) reverse transcriptase;

h”) primer, the primer structure is: 5’-Z4”-3’;

The Z4" is a sequence that can hybridize with a portion of the target RNA molecule;

The "III" system is a cDNA amplification system, and the "III" system includes:

e”) a primer having a structure of: 5’-Z1”-Z2”-Z3”-3’; and

f”) DNA polymerase;

Wherein, the Z1" is a specific recognition sequence for RNA polymerase;

Z2" is the complete or partial DR sequence;

Z3" is a sequence that partially hybridizes with the target RNA molecule or its complementary sequence;

The IV" system is a DNA transcription system, and the IV" system includes:

g”) RNA polymerase;

Preferably, when the reverse transcriptase does not have the function of digesting RNA, the "II" system further comprises RNase H enzyme;

Group B:

d”) reverse transcriptase;

h”) primer, the primer structure is: 5’-Z1”-Z2”-Z3”’-3’;

Wherein, the Z1" is a specific recognition sequence for RNA polymerase;

Z2" is the complete or partial DR sequence;

The "III" system is a cDNA amplification system, and the "III" system includes:

e") primer, the primer structure is:

5'-Z4"-3', Z4" is a sequence that can hybridize with the target RNA molecule or its complementary sequence; and

f”) DNA polymerase;

The IV" system is a DNA transcription system, and the IV" system includes:

g”) RNA polymerase;

Preferably, when the reverse transcriptase does not have the function of digesting RNA, the "II" system also includes RNase H enzyme.

5. The target nucleic acid molecule detection system according to claim 1, characterized in that the target nucleic acid molecule is a target DNA molecule, and the Cas protein cis-cleavage substrate includes a sequence identical to or complementary to the target DNA molecule; the target nucleic acid molecule detection system also includes a III"' system and a IV"' system; the III"' system and the IV"' system constitute a crRNA synthesis system; wherein

The III"' system is a DNA amplification system, and the III"' system comprises:

e"') primer, the primer structure is:

Primer 1: 5’-Z1”’-Z2”’-Z3”’-3’,

Primer 2: 5′-Z4″′-3′;

and

f”’) DNA polymerase;

Wherein, the Z1"' is a specific recognition sequence for RNA polymerase;

Z2"' is the complete or partial DR sequence;

Z3"' is a sequence that partially hybridizes with the target DNA molecule or its complementary sequence;

Z4"' is a sequence that can partially hybridize with the target DNA molecule or its complementary sequence;

The IV' system is a DNA transcription system, and the IV' system includes:

g”’) RNA polymerase.

6. The target nucleic acid molecule detection system according to claim 1, wherein the Cas protein cis-cleavage substrate is a component of the Cas protein cis-cleavage reporter molecule.

7. The target nucleic acid molecule detection system as described in claim 1 is characterized in that the Cas protein cis-cutting reporter molecule is selected from the following group: a reporter molecule based on gold nanoparticles, a reporter molecule based on fluorophores, a reporter molecule based on fluorescence polarization, a reporter molecule based on colloidal phase change/dispersion, a reporter molecule based on electrochemical signals, a reporter molecule based on semiconductor signals, and a color-based reporter molecule.

8. The target nucleic acid molecule detection system according to claim 1, characterized in that the first system further comprises:

c) A Cas protein trans-cleavage reporter molecule having a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule that does not hybridize with the guide sequence of the crRNA of the Cas protein.

9. A kit for detecting a target nucleic acid molecule, characterized in that the kit comprises:

The first kit is a kit for detecting crRNA, wherein the crRNA is prepared according to the target nucleic acid molecule, and the kit comprises:

i) a first container and a Cas protein located in the first container;

ii) a second container and a Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter molecule located in the second container, wherein the Cas protein cis-cleavage substrate or the Cas protein cis-cleavage reporter molecule comprises a sequence identical to or complementary to the sequence of the target nucleic acid molecule, and the Cas protein cis-cleavage substrate is not the target nucleic acid molecule;

iii) Optionally, a third container and a Cas protein trans-cleavage reporter molecule located in the third container, wherein the Cas protein trans-cleavage reporter molecule has a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule that does not hybridize with the guide sequence of the crRNA of the Cas protein.

10. A method for detecting a target nucleic acid molecule, characterized in that it comprises the following steps:

(i) contacting the sample to be detected with the crRNA synthesis system;

(ii) contacting the product obtained in step (i) with a crRNA detection system, wherein the crRNA detection system comprises a first system, wherein the first system comprises:

Cas proteins; and

A Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter molecule, wherein the Cas protein cis-cleavage substrate or the Cas protein cis-cleavage reporter molecule comprises a sequence identical to or complementary to the sequence of the target nucleic acid molecule, and the Cas protein cis-cleavage substrate is not the target nucleic acid molecule;

(iii) determining whether the target nucleic acid molecule is present in the sample by detecting the activation of the cleavage activity of the Cas protein.