Mutant CRISPR type V enzyme and application thereof

Abstract

The invention provides a mutant CRISPR (clustered regularly interspaced short palindromic repeats) type V enzyme and application thereof. Substrate recognition of the CRISPR enzyme variant is amplified, so that the activity of DNA (deoxyribonucleic acid) se, which is originally guided by gRNA (guide ribonucleic acid), of AaCas12b enzyme is improved, DNA is cut from original recognition DNA, and the range of efficiently recognizing DNA and cutting RNA is expanded.

Description

Technical Field

[0001] The present invention mainly relates to the field of gene editing, and specifically relates to a mutant CRISPR type V enzyme and its application. Background Art

[0002] The Type V CRISPR-Cas system is also known as the Cas12 family. It differs from other CRISPR-Cas systems in that it is an RNA-mediated single-effector ribozyme driven by a single RuvC active center. As more and more Cas12s are discovered and identified, multiple categories have been found in the family, including VA, VB, VC, VD, VE, VF, VG, VH, VI, VJ, and VK. Cas12b has excellent reaction performance and can be used in a variety of nucleic acid detection technologies combined with amplification technologies, or in clinical detection products.

[0003] In previous reports, Cas12b has RNA-mediated cis-DNase activity and trans-DNase activity, which has led to a variety of detection applications; in some reports, although Cas12a or Cas12b has RNA-mediated trans-RNase activity, the activity of trans-RNase is about 10% to 20% of trans-DNase. This one-order-of-magnitude difference in discrimination greatly limits the possibility of RNA probes as another reporting substrate for detection, and also limits the application of DNA and RNA probes as two independent substrates for reporting in a system.

[0004] In order to solve the problem of insufficient discrimination of DNA / RNA substrate recognition, the art needs to provide a new CRISPR enzyme and its application. Summary of the invention

[0005] The object of the present invention is to provide a mutated CRISPR type V enzyme and its application.

[0006] In a first aspect of the present invention, a CAS12 enzyme variant or a functional derivative thereof is provided.

[0007] The segment of the RuvC and / or Nuc active center of the wild-type CAS12 enzyme that recognizes and cuts the DNA substrate is replaced or inserted into a single-stranded nucleic acid binding domain selected from the following group: SEQ ID NO. 2, 3, 4, 5.

[0008] In another preferred example, the segment in which the RuvC and / or Nuc active centers of the wild-type CAS12 enzyme recognize and cut the DNA substrate is the 901-960 segment relative to the wild-type type V enzyme Cas12b.

[0009] In another preferred example, the wild-type CAS12 enzyme is the CRISPR type V enzyme Cas12b, and the sequence of its 919-947 segment is as shown in SEQ ID NO.1.

[0010] In another preferred example, the replacement or insertion of the segment that recognizes and cleaves the DNA substrate by the RuvC and / or Nuc active centers of the wild-type CAS12 enzyme is carried out for partial or all regions among positions 919-947 for domain replacement or insertion, where the amino acid sequence of positions 919-947 is as shown in SEQ ID NO:1.

[0011] In another preferred example, the wild-type CAS12 enzyme is AaCas12b, and the sequence is as shown in SEQ ID NO.6.

[0012] In another preferred example, the insertion of the single-stranded nucleic acid binding domain means: inserting the single-stranded nucleic acid binding domain between positions 945 and 946 of the wild-type CAS12 enzyme.

[0013] In another preferred example, the insertion of the single-stranded nucleic acid binding domain means: inserting the amino acid sequence shown in SEQ ID NO.4 or 5 between positions 945 and 946 of the wild-type CAS12 enzyme.

[0014] In another preferred example, the replacement of the single-stranded nucleic acid binding domain means: replacing positions 916-944 or 919-947 of the wild-type CAS12 enzyme with the single-stranded nucleic acid binding domain.

[0015] In another preferred example, the replacement of the single-stranded nucleic acid binding domain means: replacing positions 919-947 of the wild-type CAS12 enzyme with the amino acid sequence shown in SEQ ID NO.2.

[0016] In another preferred example, the replacement of the single-stranded nucleic acid binding domain means: replacing positions 919-947 of the wild-type CAS12 enzyme with the amino acid sequence shown in SEQ ID NO.3.

[0017] In another preferred example, the replacement of the single-stranded nucleic acid binding domain means: replacing positions 916-944 of the wild-type CAS12 enzyme with the amino acid sequence shown in SEQ ID NO.5.

[0018] In the second aspect of the present invention, there is provided a CAS12 enzyme variant or its functional derivative, which comprises one of the following mutations based on the wild-type CAS12 enzyme:

[0019] Positions 919-947 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO:2;

[0020] Positions 919-947 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO: 3;

[0021] Positions 916-944 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO: 5;

[0022] The amino acid sequence shown in SEQ ID NO. 4 or 5 is inserted between positions 945 and 946 of the wild-type CAS12 enzyme;

[0023] The amino acid sequence of the wild-type CAS12 enzyme is shown in SEQ ID NO:6.

[0024] In another preferred embodiment, the CAS12 enzyme variant is relative to the wild-type CAS12 enzyme.

[0025] Has trans-cleavage activity on DNA; and / or

[0026] RNA trans-reaction activity is increased.

[0027] In another preferred embodiment, the trans-cleavage activity C1 of the CAS12 enzyme variant or its functional derivative is increased compared to the trans-cleavage activity C0 of its wild-type CAS12 enzyme, wherein C1 / C0 ≥ 1, preferably ≥ 5, more preferably ≥ 10 or 25.

[0028] In another preferred example, the amino acid sequence of the CAS12 enzyme variant or its functional derivative is shown in any one of SEQ ID NO.7-11.

[0029] In another preferred embodiment, the CAS12 enzyme variant or its functional derivative is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs.: 7-11, an active fragment thereof, or a conservative variant polypeptide thereof.

[0030] In another preferred embodiment, the CAS12 enzyme variant or its functional derivative is selected from the following group:

[0031] (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs.: 7-11;

[0032] (b) A polypeptide derived from (a) which is obtained by replacing, deleting or adding one or more (such as 2, 3, 4 or 5) amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs.: 7-11, and has cis-cleavage activity and reduced trans-cleavage activity.

[0033] In another preferred example, the sequence of the CAS12 enzyme variant or its functional derivative is preferably as shown in SEQ ID NO.9.

[0034] In another preferred embodiment, the derived polypeptide has a homology with SEQ ID NO.: 7-11 of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%, such as 95%, 97%, 99%.

[0035] The third aspect of the present invention provides a polynucleotide encoding the CAS12 enzyme variant or its functional derivative as described in the first aspect or the second aspect of the present invention.

[0036] In another preferred embodiment, the polynucleotide encodes a polypeptide as shown in any one of SEQ ID NOs. 7-11.

[0037] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following groups on the flank of the ORF of the variant: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0038] In another preferred embodiment, the polynucleotide is selected from the following group: genomic sequence, cDNA sequence, RNA sequence, or a combination thereof.

[0039] In another preferred example, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the variant.

[0040] In another preferred embodiment, the promoter is selected from the following group: a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.

[0041] The fourth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the third aspect of the present invention.

[0042] In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selective marker, nucleic acid restriction site, and / or homologous recombination site.

[0043] In another preferred embodiment, the vector includes a plasmid vector, a phage vector, a cosmid cloning vector, a phagemid vector, an artificial chromosome vector, an episomal vector, a viral vector or a combination thereof.

[0044] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention, or the polynucleotide described in the third aspect of the present invention is integrated into its genome.

[0045] In another preferred embodiment, the host cell is a prokaryotic receptor cell.

[0046] In another preferred embodiment, further, the Escherichia coli is selected from the following group: BL21, BL21 (DE3), W3110, MG1655, RB791, RV308, HMS174, HMS174 (DE3), NM533, XL1-Blue, C600, DH1, HB101, JM109, Top10, DH5α, DH10β, TG1, BW23473, BW23474, MW003, MW005 cells or a combination thereof; the Bacillus megaterium is selected from the following group: QMB1551, PV361, DSM319 or a combination thereof.

[0047] In another preferred embodiment, the host cell is a eukaryotic cell.

[0048] In another preferred embodiment, further, the eukaryotic receptor cell is selected from the following group: yeast, fungi, plant cells, animal cells or a combination thereof.

[0049] In a sixth aspect of the present invention, a method for preparing a CAS12 enzyme variant is provided, wherein the method comprises the steps of:

[0050] (a) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for expression, thereby expressing the CAS12 enzyme variant; and

[0051] (b) isolating the CAS12 enzyme variant.

[0052] The seventh aspect of the present invention provides an enzyme preparation, which comprises the CAS12 enzyme variant described in the first aspect of the present invention or the second aspect of the present invention.

[0053] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0054] In an eighth aspect, the present invention provides a method for detecting a target nucleic acid in a sample, comprising:

[0055] Contacting the sample with the CAS12 enzyme variant or its functional derivative, the guide RNA and the target nucleic acid molecule according to the first aspect of the present invention or the second aspect of the present invention; and

[0056] The target nucleic acid molecule is detected by measuring the detectable signal generated by cleavage of the nucleic acid probe by the CAS12 enzyme variant or its functional derivative.

[0057] In another preferred embodiment, the target nucleic acid molecule is a target RNA or a target DNA.

[0058] In another preferred embodiment, the nucleic acid probe is an RNA probe or a DNA probe, and the specific composition and length of the RNA probe can be designed according to actual needs.

[0059] In another preferred embodiment, the sequence of the RNA probe is:

[0060] 5’FAM—rArArArArArArA—3’BHQ1.

[0061] In another preferred embodiment, the sequence of the DNA probe is:

[0062] 5‘HEX-TT*T*T*T*T*T-3’BHQ1;

[0063] 5‘FAM-T*T*T*T*T*TT-3’BHQ1;

[0064] 5‘FAM-C*C*C*C*C*CC-3’BHQ1

[0065] , wherein, the * mark is a thiophosphate modification.

[0066] In another preferred embodiment, the method includes contacting a sample with a nucleic acid detection composition, the nucleic acid detection composition including a Cas protein, a gRNA, and a nucleic acid probe; the gRNA including a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the nucleic acid probe, thereby detecting the target nucleic acid;

[0067] The nucleic acid detection composition is selected from one or more CAS12 enzyme variants as described in the first aspect or the second aspect of the present invention, can bind to one or more CAS12 enzyme variants as described in the first aspect or the second aspect of the present invention, a gRNA that hybridizes to a target sequence on the target nucleic acid, and a single-stranded nucleic acid probe.

[0068] In another preferred embodiment, the detection is a dual, triple, or multiplex detection.

[0069] In another preferred embodiment, the target nucleic acid includes single-stranded nucleic acid and double-stranded nucleic acid.

[0070] The ninth aspect of the present invention provides a detection system for detecting a target nucleic acid molecule, the system comprising:

[0071] The CAS12 enzyme variant or its functional derivative as described in the first aspect or the second aspect of the present invention;

[0072] A guide RNA that guides the CAS12 enzyme variant or its functional derivative to specifically bind to the target nucleic acid molecule; and a nucleic acid probe.

[0073] In another preferred example, the nucleic acid probe is a DNA nucleic acid probe or an RNA nucleic acid probe.

[0074] The tenth aspect of the present invention provides a detection system for selectively detecting a target nucleic acid molecule, the system comprising:

[0075] Two or more Cas12 proteins or functional derivatives thereof with significant differences in cleavage specificity for RNA and DNA;

[0076] A guide RNA that guides the Cas12 protein or its functional derivative to specifically bind to the target nucleic acid molecule; and

[0077] A nucleic acid probe;

[0078] Wherein at least one of the Cas12 proteins or its functional derivatives is the CAS12 enzyme variant or its functional derivative described in the first aspect or the second aspect of the present invention.

[0079] In another preferred example, at least one Cas12 protein with significant differences in cleavage specificity for RNA and DNA is any of the above-mentioned engineered CAS12 enzyme variants or their functional derivatives. Among them, the target nucleic acid molecule can be target RNA or target DNA.

[0080] In another preferred example, the nucleic acid probe is a DNA probe or an RNA probe, and the specific composition and length of the DNA probe and the RNA probe can be designed according to actual needs.

[0081] In another preferred example, the two or more Cas12 proteins or their functional derivatives can be two, three, four, or more.

[0082] In another preferred example, the detection system includes a nucleic acid detection composition, the nucleic acid detection composition includes a Cas12 protein or its functional derivative, a gRNA, and a nucleic acid probe; the gRNA includes a region that binds to the Cas12 protein or its functional derivative and a guiding sequence that hybridizes to a target sequence on the target nucleic acid; detecting a detectable signal generated by the Cas12 protein or its functional derivative cleaving the nucleic acid probe, thereby detecting the target nucleic acid.

[0083] In another preferred embodiment, the nucleic acid detection composition includes one or more CAS12 enzyme variants or functional derivatives thereof as described in the first aspect of the present invention or the second aspect of the present invention, which can be combined with one or more CAS12 enzyme variants described in the first aspect of the present invention or the second aspect of the present invention and a gRNA hybridized with a target sequence on a target nucleic acid, and a nucleic acid probe. In another preferred embodiment, the nucleic acid detection composition includes any one or both of the first nucleic acid detection composition and the second nucleic acid detection composition:

[0084] The first nucleic acid detection composition includes a first Cas12 protein, a first gRNA that binds to a CAS12 enzyme variant and hybridizes with a target sequence on a target nucleic acid, and a first nucleic acid probe;

[0085] The second nucleic acid detection composition includes a second Cas12 protein, a second gRNA that binds to a CAS12 enzyme variant and hybridizes with a target sequence on a target nucleic acid, and a second nucleic acid probe.

[0086] In another preferred example, the first Cas12 protein is a CAS12 enzyme variant, and the sequence is shown in SEQ ID NO.9 (AaCas12b-dHax).

[0087] In another preferred example, the second Cas12 protein is a second CAS12 enzyme variant, and the sequence is shown in SEQ ID NO.10 (AaCas12b+5MPL).

[0088] In another preferred embodiment, the first CAS12 enzyme variant specifically cuts the first nucleic acid probe to generate a first detectable signal;

[0089] The second CAS12 enzyme variant specifically cleaves the second nucleic acid probe, thereby generating a second detectable signal.

[0090] In another preferred embodiment, the nucleotides of the first nucleic acid probe are composed of polyT or polyA.

[0091] In another preferred example, the nucleotides of the second nucleic acid probe consist of polyT or polyA.

[0092] In another preferred embodiment, the polyT consists of 7 consecutive T bases; and / or the polyA consists of 7 consecutive A bases.

[0093] In another preferred example, the nucleic acid detection composition also includes a third Cas12 protein, a third gRNA that binds to the third Cas12 protein and hybridizes with a third target sequence on the target nucleic acid, and a third nucleic acid probe.

[0094] In another preferred embodiment, the third Cas12 protein is Cas12i.

[0095] In another preferred example, the third Cas12 protein specifically cuts the third nucleic acid probe to generate a third detectable signal.

[0096] In another preferred embodiment, the nucleotides of the third nucleic acid probe consist of polyA; the polyA consists of 7 consecutive A bases.

[0097] In another preferred embodiment, the nucleic acid probe is a DNA probe and / or an RNA probe.

[0098] In another preferred embodiment, the first detectable signal, the second detectable signal, and the third detectable signal are different detection signals from each other.

[0099] Preferably, a fluorescent group and a quenching group are respectively arranged at both ends of the nucleic acid probe, and a detectable fluorescent signal can be exhibited when the nucleic acid probe is cleaved. The fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0100] In another preferred example, the first fluorescent group and the first quenching group, the second fluorescent group and the second quenching group are respectively arranged at both ends of the first nucleic acid probe and the second nucleic acid probe; the first fluorescent group and the second fluorescent group may be the same or different fluorescent groups; the first quenching group and the second quenching group may be the same or different quenching groups.

[0101] In another preferred example, the first fluorescent group, the first quenching group, the second fluorescent group, the second quenching group, the third fluorescent group, and the third quenching group are respectively arranged at both ends of the first nucleic acid probe, the second nucleic acid probe, and the third nucleic acid probe; the first fluorescent group, the second fluorescent group, and the third fluorescent group may be the same or different fluorescent groups; the first quenching group, the second quenching group, and the third quenching group may be the same or different quenching groups.

[0102] In another preferred embodiment, different reporter groups are respectively arranged at the 5' end and the 3' end of the nucleic acid probe; or, different labeling molecules are respectively arranged at the 5' end and the 3' end of the nucleic acid probe.

[0103] In another preferred embodiment, the guide RNA refers to RNA that guides the Cas protein to specifically bind to the target DNA.

[0104] In another preferred embodiment, the detection method of the present invention can detect pathogenic microorganisms, gene mutations or specific target DNA.

[0105] In another preferred embodiment, the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, and plants; preferably, the target nucleic acid is a product enriched or amplified by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, and RAM.

[0106] In another preferred embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or a SNP site or a nucleic acid that is different from a control; preferably, the virus is a plant virus or an animal virus, for example, a papillomavirus, a hepadnavirus, a herpes virus, adenovirus, a poxvirus, a parvovirus, a coronavirus; preferably, the virus is a coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

[0107] In an eleventh aspect of the present invention, a kit for detecting a target nucleic acid in a sample is provided, the kit comprising a nucleic acid detection composition, the nucleic acid detection composition comprising a Cas protein, a gRNA and a nucleic acid probe; the gRNA comprises a region that binds to the Cas protein and a guide sequence that hybridizes with a target sequence on the target nucleic acid; a detectable signal generated by cleavage of a single-stranded nucleic acid probe by the Cas protein is detected, thereby detecting the target nucleic acid;

[0108] The nucleic acid detection composition is selected from one or more CAS12 enzyme variants as described in the first aspect of the present invention or the second aspect of the present invention, and can be combined with one or more CAS12 enzyme variants described in the first aspect of the present invention or the second aspect of the present invention and a gRNA that hybridizes with a target sequence on a target nucleic acid, and a nucleic acid probe.

[0109] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 Segments predicted to be engineerable are shown.

[0111] Figure 2The results of RNA activity test of mutants are shown. Among them, Aacas12b-PTC is the result of wild-type AaCas12b protein with target sequence as substrate, and Aacas12b-NTC is the result of wild-type AaCas12b protein with negative sample as substrate

[0112] Figure 3 Validation of the loss of trans-RNase activity of AaCas12b-SSoSSB is shown.

[0113] Figure 4 The verification of the enhanced RNase activity of AaCas12b+5MPL and AaCas12b+dHax3 is shown.

[0114] Figure 5 Verification that AaCas12b-dHax alters the base preference of DNA was shown.

[0115] Figure 6 A-6G shows different DNA binding protein domains. DETAILED DESCRIPTION

[0116] The inventors have studied extensively and deeply, and unexpectedly discovered for the first time that by protein mutation rational mutation technology, the specificity of RuvC recognition and cutting DNA substrate in Cas12b is transformed, and DNA substrate or specific recognition RNA substrate is specifically recognized, and the problem of insufficient differentiation of DNA / RNA substrate recognition is solved in practical applications. RNA probe can be used as a reporting substrate in the system, or DNA and RNA probe can be used simultaneously in a system, and two specific sgRNAs are used to guide respectively, and two or more targets are reported in parallel respectively. Then improve the reaction efficiency of RNA substrate after Cas12b trans-cutting activity is turned on, and improve the reporting efficiency of Cas12b using RNA probe. The present invention has been completed on this basis.

[0117] the term

[0118] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0119] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0120] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0121] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0122] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0123] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0124] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0125] As used herein, the "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which are derived from the immune system of microorganisms.

[0126] As used herein, "domain" or "protein domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein chain.

[0127] As used herein, "guide RNA", "sgRNA" and "gRNA" are used interchangeably herein and refer to RNA that can form a complex with Cas12 protein and target nucleic acid.

[0128] As used herein, the terms "nucleic acid", "polynucleotide" and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof and analogs thereof. "Oligonucleotide" and "oligonucleotide" are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides.

[0129] As used herein, the "complementarity" of nucleic acids refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). "Complete complementarity" refers to the formation of hydrogen bonds between all consecutive residues of a nucleic acid sequence and the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% in a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For a single base or a single nucleotide, according to the Watson-Crick base pairing principle, when A is paired with T or U, C is paired with G or I, it is called complementary or matching, and vice versa; and all other base pairings are called non-complementary or non-matching. Unless otherwise specified, the "complementary" of this application includes the cases of "completely complementary" and "substantially complementary". As long as two nucleic acid sequences can form a stable hybrid double strand through Walson-Crick base pairing, the two nucleic acid sequences are called "complementary", and the process of forming a stable hybrid double strand is called "complementary hybridization".

[0130] As used herein, the term "wild type" has a meaning generally understood by those skilled in the art, and refers to an organism, strain, gene or characteristic in a typical form that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from resources in nature and has not been intentionally modified.

[0131] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or naturally occurring.

[0132] Cas proteins

[0133] As used herein, the term "Cas12" or "Cas12 protein" or "Cas12 enzyme" includes Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, etc. In some embodiments, the Cas12 protein is a Cas12b protein, which is used in its broadest sense and includes a parent or reference Cas12b protein (e.g., AaCas12b having an amino acid sequence of SEQ ID NO: 6), derivatives or variants thereof, and functional fragments thereof, such as oligonucleotide binding fragments thereof.

[0134] The Cas protein described in the present invention is a protein having at least trans cleavage activity, preferably, the Cas protein is a protein having Cis and trans cleavage activity. The Cis activity refers to the activity of the Cas protein in recognizing the PAM site and specifically cleaving the target sequence under the action of gRNA.

[0135] Cas proteins as used herein, such as Cas12, also encompass functional variants of Cas or homologs or orthologs thereof. For example, "Cas protein variant" or "Cas protein mutant" refers to a variant or functional variant of such a protein that at least partially retains the activity of the protein. Variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs, etc. Functional variants also include fusion products of such a protein with another generally unrelated nucleic acid, protein, polypeptide or peptide. 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.

[0136] Cas12g has strong trans-cleavage activity for both ssDNA and ssRNA, and its structural analysis gives a clear structure of the zinc finger domain (Zinc Finger), which is the binding center of many transcription factors, and plays a core role in the recognition of RNA in general cognition. Cas12b and Cas12g are homologously compared, and the homologous regions of the two in the RuvC (Nuc) region are found, and the structure of the α helix in Cas12b that cooperates with RuvC and Cas12g1 homologous to the α helix is ​​identified, as well as the potential zinc ion binding domain upstream and downstream of the α helix, and it is inferred that the region is responsible for stabilizing the binding of nucleic acid substrates in Cas12b. In some embodiments, the zinc finger domain has 1 to 2 zinc binding sites, and the zinc binding site is selected from [CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC], [CxxH] One, wherein x represents any natural amino acid.

[0137] According to the crystal structure of Cas12b protein, this region is adjacent to the RuvC nucleolysis domain. By changing the binding properties of this region, the strength and specificity of nucleic acid recognition can be affected, such as the ability to recognize DNA and RNA.

[0138] As used herein, "Cas12 enzyme variant" is preferably obtained by selecting several different single-stranded nucleic acid binding domains to replace or displace the α-helix and upstream and downstream potential zinc ion binding domains (zinc finger domains) in the RuvC (Nuc) region of the original Cas12b, and obtaining a mutant that only has trans recognition activity for DNA and a mutant with improved RNA trans reaction activity.

[0139] Furthermore, by using mutants with enhanced RNA trans -reaction activity, a new method of using RNA trans -splicing activity as a reporter system was developed.

[0140] By using mutants that only have trans-recognition activity for DNA and Cas12b mutants with improved RNA trans-reaction activity, a system was developed in which Cas12b mutants can simultaneously report on two targets in the same system.

[0141] As used herein, a "functional derivative" of a protein includes various variants or functional domains of the protein. As long as the variant or functional domain retains the function of a functional domain of the protein (whether the function is enhanced or weakened), it can be called a functional derivative of the protein. For example, for Cas12 protein, Cas12 protein variants or truncations that retain the function of some of its domains are all functional derivatives of Cas12 protein.

[0142] Nucleic acid probe

[0143] The nucleic acid probe or single-stranded nucleic acid probe of the present invention includes different reporter groups or labeling molecules at both ends, and does not present a reporter signal when it is in the initial state (i.e., not cut state), and presents a detectable signal after the single-stranded nucleic acid is cut, i.e., a detectable difference is present after cutting and before cutting. In the present invention, if a detectable difference can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or, if the detectable difference cannot be detected, it reflects that the target nucleic acid does not contain the characteristic sequence to be detected.

[0144] In one embodiment, the reporter group or labeling molecule includes a fluorescent group and a quencher group, the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quencher group is selected from one or any several of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0145] Target nucleic acid

[0146] As used herein, the term "target nucleic acid", "target nucleic acid" or "target nucleic acid molecule" refers to the target nucleic acid in a sample, which may be a target RNA or a target DNA, or may contain both target RNA and target DNA.

[0147] In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or a SNP site or a nucleic acid that differs from a control; preferably, the virus is a plant virus or an animal virus, for example, a papillomavirus, a hepadnavirus, a herpes virus, an adenovirus, a poxvirus, a parvovirus, a coronavirus; preferably, the virus is a coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

[0148] In some embodiments, the target nucleic acid is derived from cells, for example, from cell lysate.

[0149] In some embodiments, the measurement of the detectable signal can be quantitative, and in other embodiments, the measurement of the detectable signal can be qualitative.

[0150] Variants

[0151] As used herein, "variant" or "mutant" is interpreted as a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, respectively, but retains the necessary characteristics. A typical variant of a polynucleotide is different from the nucleic acid sequence of another reference polynucleotide. Changes in the variant nucleic acid sequence may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide is different from another reference polypeptide in amino acid sequence. Typically, the difference is limited, so that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by any combination of one or more substitutions, additions, deletions. The amino acid residues replaced or inserted may or may not be amino acid residues encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring (such as an allelic variant), or may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0152] CAS12 enzyme variants of the present invention also include active fragments, derivatives and analogs thereof. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of CAS12 enzyme variants of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) polypeptides having substitution groups in one or more amino acid residues, or (iii) polypeptides formed by fusion with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) additional amino acid sequences fused to this polypeptide sequence to form a polypeptide (a fusion protein formed by fusion with a tag sequence such as a leader sequence, a secretory sequence or 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the well-known range of those skilled in the art.

[0153] A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0154] Table A Conservative variant polypeptide amino acid substitution table

[0155]

[0156]

[0157] The present invention also provides analogs of the CAS12 enzyme variants of the present invention. These analogs may differ from the polypeptides of the present invention either in terms of amino acid sequence differences, or in terms of modified forms that do not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0158] In addition, the CAS12 enzyme variants of the present invention can also be modified. Modified forms (which generally do not change the primary structure) include: chemically derivatized forms of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides that are glycosylated during the synthesis and processing of the polypeptide or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that effect glycosylation (such as mammalian glycosylating or deglycosylating enzymes). Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to enhance their proteolytic resistance or to optimize their solubility properties.

[0159] The term "polynucleotide encoding a CAS12 enzyme variant of the present invention" may be a polynucleotide that includes the polynucleotide encoding a CAS12 enzyme variant of the present invention, or may also be a polynucleotide that further includes additional coding and / or non-coding sequences.

[0160] The present invention also relates to variants of the above polynucleotides that encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs, and derivatives of the CAS12 enzyme variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide that may be substitutions, deletions, or insertions of one or more nucleotides, but that do not substantially alter the function of the encoded CAS12 enzyme variant.

[0161] The present invention also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" mean: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more.

[0162] The CAS12 enzyme variants and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0163] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0164] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0165] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0166] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0167] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0168] Expression vector

[0169] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the CAS12 enzyme variant of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0170] By conventional recombinant DNA techniques, the polynucleotide sequence of the present invention can be used to express or produce recombinant CAS12 enzyme variants. Generally speaking, the following steps are included:

[0171] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a CAS12 enzyme variant of the present invention, or a recombinant expression vector containing the polynucleotide;

[0172] (2) Host cells cultured in a suitable culture medium;

[0173] (3) Isolate and purify proteins from culture medium or cells.

[0174] In the present invention, the polynucleotide sequence encoding the CAS12 enzyme variant can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and stabilize in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0175] Methods well known to those skilled in the art can be used to construct expression vectors containing CAS12 enzyme variant encoding DNA sequences of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to a suitable promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation.

[0176] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0177] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0178] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0179] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0180] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0181] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0182] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0183] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0184] application

[0185] The present application also provides a detection system for detecting a target nucleic acid molecule, the system comprising: any one or more of the above-mentioned CAS12 enzyme variants or their functional derivatives; a guide RNA, wherein the guide RNA guides the Cas12 protein or its functional derivative to specifically bind to the target nucleic acid molecule; and a nucleic acid probe. Among them, the target nucleic acid molecule can be a target RNA or a target DNA. In some embodiments, the nucleic acid probe is an RNA probe, and the specific composition and length of the RNA probe can be designed according to actual needs.

[0186] The present application also provides a method for detecting a target nucleic acid molecule in a sample, comprising: contacting the sample with any one or more of the above-mentioned CAS12 enzyme variants or their functional derivatives, guide RNA and target nucleic acid molecules; and measuring a detectable signal generated by cleavage of a nucleic acid probe by the CAS12 enzyme variant or its functional derivative, thereby detecting the target nucleic acid molecule. Wherein, the target nucleic acid molecule can be a target RNA or a target DNA.

[0187] The present application also provides a detection system for selectively detecting a target nucleic acid molecule, the system comprising: two or more Cas12 proteins or their functional derivatives having significant differences in cutting specificity for RNA and DNA; guide RNA, the guide RNA guiding the Cas12 protein or its functional derivative to specifically bind to the target nucleic acid molecule; and a nucleic acid probe. Among them, the two or more Cas12 proteins or their functional derivatives can be two, three, four, or more.

[0188] In some embodiments, at least one Cas12 protein or its functional derivative having significant differences in the cutting specificity of RNA and DNA is any of the above-mentioned CAS12 enzyme variants or its functional derivatives. Wherein, the target nucleic acid molecule can be a target RNA or a target DNA.

[0189] In some embodiments, the nucleic acid probe is a DNA probe or an RNA probe, and the specific composition and length of the DNA probe and the RNA probe can be designed according to actual needs.

[0190] The main advantages of the present invention include

[0191] (1) The modifiable region of Cas12b identified by the inventors can be extended by replacing or inserting different single-stranded binding proteins, and combining changes, so that the substrate recognition of the AaCas12b enzyme is extended, and the original DNAse activity guided by gRNA of the AaCas12b enzyme is improved, from the original recognition of DNA cutting DNA to the range of efficiently recognizing DNA cutting RNA.

[0192] (2) The mutant protein of the present invention can be used for in vitro detection applications. In addition, the mutant enzyme provided by the present invention can specifically recognize DNA probes without cutting RNA substrates after being activated; the mutant enzyme provided by the present invention can efficiently use RNA probes after being activated, thereby serving as a reporting system independent of the previous enzyme.

[0193] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0194] Example 1 Sequence alignment and mutation region determination of Cas12b and Cas12g

[0195] By using the CLUSTAL 2.1 Multiple Sequence Alignments tool, Cas12b and Cas12g were aligned homologously, and it was found that the two had a high similarity in the RuvC (Nuv) region. The structure of the α-helix in Cas12b that cooperates with RuvC and is homologous to Cas12g1 was identified, as well as the potential sites P916 and L941 ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) upstream and downstream of the α-helix that can form stronger zinc binding; combined with the crystal structure analysis of Cas12b, it is speculated that there may be gate points F897 and F964 upstream and downstream that restrict the stable entry of larger structure ribose, and these structures are hidden in the 882-992 RuvC domain.

[0196]

[0197]

[0198] like Figure 1 The predicted segment that can be engineered is P916 to P945.

[0199] Example 2 Structural mutation of the α-helix upstream and downstream of the gatekeeper amino acid of Cas12b and protein acquisition

[0200] For the segments predicted to be editable by Cas12b, the estimated features are DNA recognition and grabbing. Several structures of nucleic acid binding proteins were selected, including single-stranded binding proteins in prokaryotes, to mutate the wild-type AaCas12b in the following 9 ways. The results of ClustalW alignment after mutation are shown below:

[0201] Alignment of substitution mutations in DNA binding protein domains

[0202]

[0203]

[0204] Comparison of insertion mutations in DNA- and RNA-binding protein domains

[0205]

[0206] (1) The 919-947 segment of the amino acid sequence of wild-type AaCas12b (the amino acid sequence of this segment is CAREQNPEPFPWWLNKFVAEHKLDGCPLR, SEQ ID NO.1),

[0207] Replaced with a segment of the TthSSB protein (the amino acid sequence of the segment is

[0208] TAVARLGLAVNERRQGAEERTHFVEVQAWRDLAEWAAELRKGDGLFVIGRLVNDSWTSSSGERRFQTRVEALRLERPTR, SEQ ID NO.2), the resulting protein is called AaCas12b-TthSSB, and its amino acid sequence is shown in SEQ ID NO: 7. The underlined part represents the replaced segment.

[0209] (2) The 919-947 segment of the amino acid sequence of the wild-type AaCas12b was replaced with the segment of the SsoSSB protein:

[0210] TVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIA, SEQ ID NO.3), the resulting protein is called AaCas12b-SSoSSB, and its amino acid sequence is shown in SEQ ID NO: 6. The underlined part represents the replaced segment.

[0211] (3) The 916-944 segment of the amino acid sequence of the wild-type AaCas12b is replaced with the effector monomer of the TALE protein (the amino acid sequence of this segment is GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN, SEQ ID NO.5), and the resulting protein is called AaCas12b-dHax, and its amino acid sequence is shown in SEQ ID NO: 15. The underlined portion represents the replaced segment.

[0212] (4) Insert a segment of 5MPL protein after position 945 (between positions 945 and 946) of the amino acid sequence of wild-type AaCas12b (the amino acid sequence of this segment is:

[0213] GGGSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRGGGSP, SEQ ID NO.4), the resulting protein is called AaCas12b+5MPL, and its amino acid sequence is shown in SEQ ID NO: 10. The underlined portion represents the inserted segment.

[0214] (5) Insert 3x TALE effector monomers after position 945 (between positions 945 and 946) of the amino acid sequence of wild-type AaCas12b (the amino acid sequence of this segment is:

[0215] GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN, SEQ ID NO.5), the resulting protein is called AaCas12b+dHax3, and its amino acid sequence is shown in SEQ ID NO: 9. The underlined part represents the inserted segment.

[0216] The corresponding vectors were designed according to the amino acid sequences of the above five mutants, and the vectors were transferred into the pET28 expression vector and induced to express in the Escherichia coli strain BL21. Five corresponding mutant proteins were obtained by protein purification methods such as affinity chromatography and ion exchange chromatography.

[0217] Example 3: Preliminary determination of the activity of mutants

[0218] The final concentration of the activity assay system is: 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ulCas12b protein, 500nM probe, as shown in the table, 42°C, reaction for 1h. The HEX channel fluorescence is collected on the Macrostone SLAN 96 instrument, and the final results of DNA activity assay are displayed with the starting fluorescence value, and the RNA activity test results are displayed with the final fluorescence value.

[0219] Table B DNA activity assay system and related sequences of different protein mutants

[0220] Component name Use concentration Final concentration 1rxn / ul <![CDATA[ddH 2 The]]> / / 4 rCutSmart 10X 1X 2 Rnase inhibitor 20 U / μL 0.5U / μL 0.5 Cas12b protein mutants 600ng / ul 60ng / ul 2 <![CDATA[sgRNA 2 ]]> 300ng / ul 7.5ng / ul 0.5 <![CDATA[Probe 1 ]]> 5μM 500nM 2 <![CDATA[Sample 2 > 2E11copies / ul 1E12copies 5

[0221] 1. Probe sequence information

[0222] DNA or RNA probes sequence T-FAM-BHQ1 5'FAM-TTTTTTT-3'BHQ1 C-FAM-BHQ1 5'FAM-CCCCCCC-3'BHQ1 A-FAM-BHQ1 5'FAM—AAAAAAA—3'BHQ1 G-FAM-BHQ1 5'FAM—GGGGGGG—3'BHQ1 rA-RNA-BHQ1 5'FAM—rArArArArArArA—3'BHQ1 rC-RNA-BHQ1 5'FAM—rCrCrCrCrCrCrC—3'BHQ1 rU-RNA-BHQ1 5'FAM—rUrUrUrUrUrUrU—3'BHQ1 rG-RNA-BHQ1 5'FAM—rGrGrGrGrGrGrG—3'BHQ1

[0223] 2. sgRNA and template sequence information

[0224]

[0225]

[0226] like Figure 2 , we can see that the mutant has the following changes:

[0227] a. AaCas12b-TthSSB lost its trans-cleavage activity for DNA and RNA after replacing the original binding region;

[0228] b. After replacing the original binding region, AaCas12b-SSoSSB lost the trans-cleavage activity of RNA, retained the trans-cleavage activity of DNA, changed the editing preference of the original DNA trans-cleavage activity, and became incompatible with the A base;

[0229] c. After AaCas12b-dHax replaced the original binding region, the base preference was changed, and the trans-cleavage activity of DNA became more favorable to the T base, and had no affinity for the A and C bases;

[0230] d. AaCas12b+5MPL retained the trans-cleavage activity of DNA and obtained higher trans-cleavage activity of RNA;

[0231] e. AaCas12b+dHax3 retained the trans-cleavage activity of DNA and obtained higher trans-cleavage activity of RNA;

[0232] Example 4: Verification of the loss of trans-RNase activity of AaCas12b-SSoSSB

[0233] AaCas12bSSoSSB verifies the cleavage system;

[0234] The final concentration of the activity assay system was 1xrCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul Cas12b protein, 500nM RNA probe, as shown in Table C, 42°C, reaction for 1h. In addition to the above, additional ions MnCl 2 , CaCl 2 、CoCl 2 , Spermidine, 42°C, react for 1 hour. The fluorescence of HEX channel was collected on the Macrostone SLAN 96 instrument, and the maximum value of fluorescence increase per unit time was used to calculate the final result.

[0235] Table C AaCas12b-SSoSSB RNA cleavage activity verification system under different cofactors

[0236]

[0237] 1. Probe details

[0238] sequence rA-RNA-BHQ1 5'FAM—rArArArArArArA—3'BHQ1

[0239] 2. sgRNA and template sequence information

[0240]

[0241] As above Figure 3 It can be seen that AaCas12b-SSoSSB cannot activate the trans-RNase activity under the conditions of various additives.

[0242] Example 5: Verification of RNase activity enhancement of AaCas12b+5MPL and AaCas12b+dHax3

[0243] AaCas12b+5MPL, AaCas12b+dHax3 verified the cleavage system;

[0244] The final concentration of the activity assay system was 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul Cas12b protein, 500nM RNA probe, as shown in Table D, 42°C, and reacted for 1h. The HEX channel fluorescence was collected on the Macrostone SLAN 96 instrument, and the maximum value of the fluorescence increase per unit time was used to calculate the final result.

[0245] Table D AaCas12b+5MPL, AaCas12b+dHax3 RNA cleavage activity verification system

[0246] Component name Use concentration Final concentration 1rxn / ul ddH2O / / To 20ul rCutSmart 10X 1X 2 Rnase inhibitor 20 U / μL 0.5U / μL 0.5 Cab12 protein mutants 600ng / ul 60ng / ul 2 <![CDATA[sgRNA 2 ]]> 300ng / ul 7.5ng / ul 0.5 <![CDATA[Probe 1 ]]> 5μM 500nM 2 <![CDATA[Sample 2 > 2E11copies / ul 1E12copies 5

[0247] 1. Probe details

[0248] sequence rA-RNA-BHQ1 5'FAM—rArArArArArArA—3'BHQ1

[0249] 2. sgRNA and template sequence information

[0250]

[0251] like Figure 4 As shown, 1, 2, and 3 are comparisons of the trans and RNase activities of AaCas12b+5MPL, AaCas12b+dHax3, and wild-type AaCas12b.

[0252] Example 6: Verification of AaCas12b-dHax changes the base preference of DNA

[0253] The final concentrations of reagents added to the cleavage reaction system for the three proteins AaCas12b, AaCas12b-dHax, and AaCas12b-SSoSSB were: 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul Cas12b protein, template, and 500nM DNA probes with two different positions of thiolation and different fluorescent groups, at 42°C for 1h. The fluorescence of the HEX channel was collected on the Macrostone SLAN 96 instrument, and the maximum value of the fluorescence increase per unit time was used to calculate the final results.

[0254] Table E AaCas12b-dHax cleavage preference verification system

[0255] Component name Use concentration Final concentration 1rxn / ul <![CDATA[ddH 2 The]]> / / To 20ul rCutSmart 10X 1X 2 Rnase inhibitor 20 U / μL 0.5U / μL 0.5 Cab12 protein mutants 600ng / ul 60ng / ul 2 <![CDATA[sgRNA 2 ]]> 300ng / ul 7.5ng / ul 0.5 <![CDATA[Probe 1 ]]> 5μM 500nM 2 <![CDATA[Sample 2 > 2E11copies / ul 1E12copies 5

[0256] 1. Probe details

[0257] Probe sequence HEX-DNA-probe 5'HEX-TT*T*T*T*T*T*T-3'BHQ1 FAM-DNA-probe 5'FAM-C*C*C*C*C*CC-3'BHQ1

[0258] 2. sgRNA and template sequence information

[0259]

[0260] like Figure 5 What is shown is that after AaCas12b-dHax replaced the original nucleic acid domain, the activity ratio of dT and dC changed.

[0261] Example 7: Using AaCas12b+5MPL, AaCas12b+dHax3, Cas12i, and using RNA probes for detection

[0262] AaCas12b+5MPL: in 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul AaCas12b+5MPL protein, novel coronavirus sample, 500nM RNA probe, 42°C, reaction for 1h.

[0263] AaCas12b+dHax3: in 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul AaCas12b+dHax3 protein, novel coronavirus sample, 500nM RNA probe, 42°C, reaction for 1h.

[0264] Cas12i: in 1x rCutSmart buffer, 7.5ng / ul sgRNA, 60ng / ul Cas12i protein, novel coronavirus sample, 500nM RNA probe, 42°C, reaction for 1h.

[0265] The results showed that the above three proteins AaCas12b+5MPL, AaCas12b+dHax3, and Cas12i can successfully detect new coronavirus positive samples.

[0266] Example 8: Using Cas12b-dHax and Cas12i, DNA probes are used for dual detection in one system

[0267] Cas12b-dHax and Cas12i use two DNA probes with different thiolation positions and different fluorescent groups and are added to the same system. The Cas12b-dHax DNA probe is FAM fluorescent, and Cas12i is HEX fluorescent.

[0268] Table F DNA probe dual detection system

[0269] Component name Use concentration Final concentration 1rxn / ul <![CDATA[ddH 2 The]]> / / To 20ul rCutSmart 10X 1X 2 Rnase inhibitor 20 U / μL 0.5U / μL 0.5 Cas12b-dHax 600ng / ul 60ng / ul 1 Cas12b-dHax sgRNA 300ng / ul 7.5ng / ul 0.5 Cas12i 600ng / ul 60ng / ul 1 Cas12i sgRNA 300ng / ul 7.5ng / ul 0.5 <![CDATA[HEX-DNA-probe 1 ]]> 5μM 500nM 2 <![CDATA[FAM-DNA-probe 1 ]]> 5μM 500nM 2 Novel coronavirus samples 2.5 HPV Sample 2.5

[0270] 1. Probe information

[0271] sequence HEX-DNA-probe 5'HEX-TT*T*T*T*T*T*T-3'BHQ1 FAM-DNA-probe 5'FAM-C*C*C*C*C*CC-3'BHQ1

[0272] The results showed that in the same reaction system, different fluorescence channels of the new coronavirus samples and HPV samples could collect corresponding fluorescence signals, and positive samples could be successfully detected.

[0273] Example 9: Using Cas12b-dHax and Cas12i, RNA probes are used for dual detection in one system

[0274] Cas12b-dHax and Cas12i use two RNA probes with different thiolation positions and different fluorescent groups and are added to the same system. The Cas12b-dHax RNA probe is FAM-RNA-probe, and the Cas12i is HEX-RNA-probe.

[0275] Table G RNA probe dual detection system

[0276] Component name Use concentration Final concentration 1rxn / ul <![CDATA[ddH 2 The]]> / / To 20ul rCutSmart 10X 1X 2 Rnase inhibitor 20 U / μL 0.5U / μL 0.5 Cas12b-dHax 600ng / ul 60ng / ul 1 Cas12b-dHax sgRNA 300ng / ul 7.5ng / ul 0.5 Cas12i 600ng / ul 60ng / ul 1 Cas12i sgRNA 300ng / ul 7.5ng / ul 0.5 <![CDATA[HEX-RNA-probe 1 ]]> 5μM 500nM 2 <![CDATA[FAM-RNA-probe 1 ]]> 5μM 500nM 2 Novel coronavirus samples 2.5 HPV Sample 2.5

[0277] 1. Probe information

[0278] sequence HEX-RNA-probe 5'HEX-rArA*rA*rA*rA*rA*rA-3'BHQ1 FAM-RNA-probe 5'FAM-rA*rA*rA*rA*rA*rArA-3'BHQ1

[0279] The results showed that in the same reaction system, different fluorescence channels of the new coronavirus samples and HPV samples could collect corresponding fluorescence signals, and positive samples could be successfully detected.

[0280] discuss:

[0281] The application utilizes RuvC and NuC regions in Cas12b to improve the specificity of identifying and cutting DNA substrates. By replacing or inserting different single-stranded binding proteins or combinations thereof, such as different nucleic acid binding proteins (segments of TthSSB protein, segments of SsoSSB protein, segments of 5MPL protein, TALE effector concatemers), different Cab12 mutants are obtained. The Cab12 mutant of the present invention expands the substrate recognition of Cas12b enzyme, can specifically identify DNA substrates or RNA substrates (i.e., single-stranded nucleic acid probes), and the transformation method of the present invention can be used for the performance transformation of other Cas12 enzymes containing NuC regions. Through the above transformation, it is realized in the same system, using the mutant of the Cas12b of the present invention to carry out dual or multiple detections simultaneously.

[0282] Sequence of the present invention:

[0283] SEQ ID NO.1 Segment 919-947 of the amino acid sequence of wild-type AaCas12b

[0284] CAREQNPEPFPWWLNKFVAEHKLDGCPLR

[0285] SEQ ID NO.2 Segment of TthSSB protein

[0286] TAVARLGLAVNERRQGAEERTHFVEVQAWRDLAEWAAELRKGDGLFVIGRLVNDSWTSSSSGERRFQTRVEALRLERPTR

[0287] SEQ ID NO.3 Segment of SsoSSB protein

[0288] TVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIA

[0289] SEQ ID NO.4 Segment of 5MPL protein

[0290] GGGSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRGGGSP

[0291] SEQ ID NO.5 3x TALE effector monomer

[0292] GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN

[0293] SEQ ID NO:6>WT AapCas12b

[0294]

[0295] SEQ ID NO.7 AaCas12b-TthSSB

[0296]

[0297] SEQ ID NO.8 AaCas12b-SSoSSB

[0298]

[0299] SEQ ID NO.9 AaCas12b-dHax

[0300]

[0301] SEQ ID NO.10 AaCas12b+5MPL

[0302]

[0303] SEQ ID NO.11 AaCas12b+dHax3

[0304] MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCP GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNG GKQALETVQRLLPVLCQAHGLPPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

[0305] SEQ ID NO:20>WT Cas12g1

[0306] MKIEEGKGHHHHHHMAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQWAIQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPAFTNELRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNYEEWKKEKQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKHSVLCNQFNEFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEETLRGKNGGRLPHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRKPVFRYPSVKRHSIAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGETEISSVHLHFVGTRPRIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLLETFPGNAEQELRLLAVDLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRDASSKQPRPGLSRDHVGRHLQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRGLTVHTARMIRDWARLNARQIIQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHGRIRRKVTEKAVERGMRVVTVPYLASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQVDENAARVLGRVFWGEIELPTAIP

[0307] SEQ ID NO:21 Cas12b-ggg

[0308] MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPAR GGGGGGPLRADDLIPTGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI

[0309] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A CAS12 enzyme variant or a functional derivative thereof, It is characterized in that The segment of the RuvC and / or Nuc active center of the wild-type CAS12 enzyme that recognizes and cuts the DNA substrate is replaced or inserted into a single-stranded nucleic acid binding domain selected from the following group: SEQ ID NO.2, 3, 4, 5.

2. The CAS12 enzyme variant or a functional derivative thereof as claimed in claim 1, It is characterized in that The replacement or insertion of the segment of the RuvC and / or Nuc active center of the wild-type CAS12 enzyme that recognizes and cleaves the DNA substrate is to replace or insert the domain in part or all of the region at positions 919-947, wherein the amino acid sequence at positions 919-947 is shown in SEQ ID NO:

1.

3. The CAS12 enzyme variant or a functional derivative thereof as claimed in claim 1, It is characterized in that It comprises one of the following mutations based on the wild-type CAS12 enzyme: Positions 919-947 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO: 2; Positions 919-947 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO: 3; Positions 916-944 of the wild-type CAS12 enzyme are replaced with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO.4 or 5 is inserted between positions 945 and 946 of the wild-type CAS12 enzyme; wherein the amino acid sequence of the wild-type CAS12 enzyme is shown in SEQ ID NO:

6.

4. The CAS12 enzyme variant or a functional derivative thereof as claimed in claim 3, It is characterized in that The CAS12 enzyme variant or its functional derivative is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs.: 7-11, an active fragment thereof, or a conservative variant polypeptide thereof.

5. A polynucleotide, It is characterized in that The polynucleotide encodes a CAS12 enzyme variant or a functional derivative thereof as described in any one of claims 1 to 4.

6. A carrier, It is characterized in that The vector contains the polynucleotide according to claim 5.

7. A host cell, It is characterized in that The host cell contains the vector according to claim 6, or the polynucleotide according to claim 5 is integrated into its genome.

8. A method for preparing a CAS12 enzyme variant, It is characterized in that The method comprises the steps of: (a) culturing the host cell according to claim 7 under conditions suitable for expression, thereby expressing the CAS12 enzyme variant; and (b) isolating the CAS12 enzyme variant.

9. A method for detecting a target nucleic acid in a sample, It is characterized in that include Contacting the sample with a CAS12 enzyme variant or a functional derivative thereof as described in any one of claims 1 to 4, a guide RNA and a target nucleic acid molecule; and The target nucleic acid molecule is detected by measuring the detectable signal generated by cleavage of the nucleic acid probe by the CAS12 enzyme variant or its functional derivative.

10. A detection system for detecting a target nucleic acid molecule, the system comprising: The CAS12 enzyme variant or its functional derivative according to any one of claims 1 to 4; A guide RNA, wherein the guide RNA guides the CAS12 enzyme variant or its functional derivative to specifically bind to a target nucleic acid molecule; and a nucleic acid probe.

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