CN120026005A - Mutated CRISPR type V enzyme and its application - Google Patents

Abstract

The present invention provides a mutant CRISPR type V enzyme and its application. The substrate recognition of the CRISPR enzyme variant of the present invention is amplified, so that the DNAse activity of the AaCas12b enzyme originally guided by gRNA is improved, and the scope of efficiently recognizing DNA and cutting RNA is expanded from the original recognition of DNA to cutting DNA.

Description

Mutant CRISPR TYPEV enzymes and uses thereof

Technical Field

The present invention relates generally to the field of gene editing. In particular, it relates to mutant CRISPR TYPEV enzymes and their use.

Background

TYPE V CRISPR-Cas system, also known as Cas12 family, differs from other CRISPR-Cas systems in that it is a single-effect daughter ribozyme mediated by RNA driven by a single RuvC active center. As more and more Cas12 is mined and identified, it has been found that there are multiple categories of V-A, V-B, V-C, V-D, V-E, V-F, V-G, V-H, V-I, V-J, V-K, etc. in this family. Cas12b has excellent reactivity and can be used in a variety of nucleic acid detection techniques in combination with amplification techniques, or in clinical detection products.

In the following reports, cas12b has RNA-mediated cis-DNase activity and trans-DNase activity, so that various detection applications have been developed, and in some reports, cas12a or Cas12b has RNA-mediated trans-RNase activity, but the activity of trans-RNase is about 10% -20% of that of trans-Dnase. This difference in discrimination by an order of magnitude greatly limits the possibility of detection of the RNA probe as an alternative reporter substrate and also limits the use of DNA and RNA probes for reporting on separate substrates in a single system.

In order to solve the problem of insufficient discrimination of DNA/RNA substrate recognition, there is a need in the art to provide a novel CRISPR enzyme and its use.

Disclosure of Invention

The invention aims at providing a mutated CRISPR TYPEV enzyme and application thereof.

In a first aspect of the invention, there is provided a CAS12 enzyme variant or functional derivative thereof,

The RuvC and/or Nuc active center recognition cleavage DNA substrate segment of the wild-type CAS12 enzyme is substituted or inserted into a single-stranded nucleic acid binding domain selected from the group consisting of SEQ ID nos. 2, 3, 4, 5.

In another preferred embodiment, the RuvC and/or Nuc active center recognition cleavage DNA substrate segment of the wild-type CAS12 enzyme is a 901-960 segment relative to the wild-type typeV enzyme CAS12 b.

In another preferred embodiment, the wild-type CAS12 enzyme is CRISPR TYPEV enzyme Cas12b, the sequence of the 919-947 segment of which is shown in SEQ ID NO. 1.

In another preferred embodiment, the substitution or insertion of the segment of the wild-type CAS12 enzyme that recognizes cleavage DNA substrates for ruvC and/or Nuc is a domain substitution or insertion for part or all of the region in positions 919-947, wherein the amino acid sequence at positions 919-947 is shown in SEQ ID NO. 1.

In another preferred embodiment, the wild-type CAS12 enzyme is AaCas b, the sequence of which is shown in SEQ ID NO. 6.

In another preferred embodiment, the insertion of the single stranded nucleic acid binding domain refers to insertion of the single stranded nucleic acid binding domain between positions 945 and 946 of the wild-type CAS12 enzyme.

In another preferred embodiment, the insertion of the single stranded nucleic acid binding domain refers to insertion of the amino acid sequence shown in SEQ ID NO.4 or 5 between positions 945 and 946 of the wild-type CAS12 enzyme.

In another preferred embodiment, the replacement of the single-stranded nucleic acid binding domain refers to replacement of the single-stranded nucleic acid binding domain at positions 916-944 or 919-947 of the wild-type CAS12 enzyme.

In another preferred embodiment, the replacement of the single-stranded nucleic acid binding domain refers to the replacement of the amino acid sequence shown in SEQ ID NO.2 at positions 919-947 of the wild-type CAS12 enzyme.

In another preferred embodiment, the replacement of the single-stranded nucleic acid binding domain refers to the replacement of the amino acid sequence shown in SEQ ID NO.3 at positions 919-947 of the wild-type CAS12 enzyme.

In another preferred embodiment, the replacement of the single-stranded nucleic acid binding domain refers to the replacement of the amino acid sequence shown in SEQ ID NO.5 at positions 916-944 of the wild-type CAS12 enzyme.

In a second aspect of the invention, there is provided a CAS12 enzyme variant or functional derivative thereof comprising one of the following mutations based on a wild-type CAS12 enzyme:

the 919-947 position of the wild CAS12 enzyme is replaced by the amino acid sequence shown as SEQ ID NO. 2;

the 919-947 position of the wild CAS12 enzyme is replaced by the amino acid sequence shown in SEQ ID NO. 3;

the 916-944 position of the wild CAS12 enzyme is replaced by 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 945 and 946 of the wild-type CAS12 enzyme;

Wherein the amino acid sequence of the wild CAS12 enzyme is shown as SEQ ID NO. 6.

In another preferred embodiment, the CAS12 enzyme variant is a variant of a wild type CAS12 enzyme,

Has trans-cleavage activity on DNA, and/or

The RNA trans-reactivity is improved.

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

In another preferred embodiment, the amino acid sequence of the CAS12 enzyme variant or functional derivative thereof is as shown in any one of SEQ ID NO. 7-11.

In another preferred embodiment, the CAS12 enzyme variant or functional derivative thereof is a polypeptide having the amino acid sequence shown in any one of SEQ ID No. 7-11, an active fragment thereof, or a conservatively variant polypeptide thereof.

In another preferred embodiment, the CAS12 enzyme variant or functional derivative thereof is selected from the group consisting of:

(a) A polypeptide having an amino acid sequence as set forth in any one of SEQ ID nos. 7 to 11;

(b) A polypeptide derived from (a) having cis-cleavage activity and reduced trans-cleavage activity, wherein the amino acid sequence as set forth in any one of SEQ ID nos 7 to 11 is formed by substitution, deletion or addition of one or more (e.g. 2, 3, 4 or 5) amino acid residues.

In another preferred embodiment, the CAS12 enzyme variant or functional derivative thereof has a sequence as shown in SEQ ID NO. 9.

In another preferred embodiment, the derivatized polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, such as 95%, 97%, 99% homology to the sequence of SEQ ID No. 7-11.

In a third aspect of the invention, there is provided a polynucleotide encoding a CAS12 enzyme variant or a functional derivative thereof according to the first or second aspect of the invention.

In another preferred embodiment, the polynucleotide encodes a polypeptide as set forth in any one of SEQ ID NO. 7-11.

In another preferred embodiment, the polynucleotide additionally comprises an auxiliary element selected from the group consisting of a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof, flanking the ORF of the variant.

In another preferred embodiment, the polynucleotide is selected from the group consisting of genomic sequences, cDNA sequences, RNA sequences, or combinations thereof.

In another preferred embodiment, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the variant.

In another preferred embodiment, the promoter is selected from the group consisting of a constitutive promoter, a tissue specific promoter, an inducible promoter, or a strong promoter.

In a fourth aspect of the invention there is provided a vector comprising a polynucleotide according to the third aspect of the invention.

In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the vector comprises 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.

In a fifth aspect of the invention there is provided a host cell comprising a vector according to the fourth aspect of the invention, or having integrated into its genome a polynucleotide according to the third aspect of the invention.

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

In another preferred embodiment, the E.coli is selected from the 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 and the B.megaterium (Bacillus megaterium) is selected from the group QMB1551, PV361, DSM319 or a combination thereof.

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

In another preferred embodiment, further, the eukaryotic recipient cell is selected from the group consisting of a yeast, a fungus, a plant cell, an animal cell, or a combination thereof.

In a sixth aspect of the present invention, there is provided a method for preparing a CAS12 enzyme variant, the method comprising the steps of:

(a) Culturing the host cell of the fifth aspect of the invention under conditions suitable for expression, thereby expressing the CAS12 enzyme variant, and

(B) Isolating the CAS12 enzyme variant.

In a seventh aspect of the invention, an enzyme preparation is provided, comprising the CAS12 enzyme variant of the first aspect of the invention or the second aspect of the invention.

In another preferred embodiment, the enzyme preparation comprises an injectable preparation, and/or a lyophilized preparation.

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

Contacting the sample with a CAS12 enzyme variant or functional derivative thereof, a guide RNA, and a target nucleic acid molecule according to the first aspect of the invention or the second aspect of the invention, and

Measuring a detectable signal generated by cleavage of a nucleic acid probe by the CAS12 enzyme variant or functional derivative thereof, thereby detecting the target nucleic acid molecule.

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

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 may be designed according to actual needs.

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

5’FAM—rArArArArArArA—3’BHQ1。

in another preferred embodiment, the DNA probe has the sequence:

5‘HEX-TT*T*T*T*T*T-3’BHQ1;

5‘FAM-T*T*T*T*T*TT-3’BHQ1;

5‘FAM-C*C*C*C*C*CC-3’BHQ1

Wherein the label is a thio modification.

In another preferred embodiment, the method comprises contacting the sample with a nucleic acid detection composition comprising a Cas protein, a gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on a target nucleic acid, and a nucleic acid probe;

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

In another preferred embodiment, the assay is a dual, triple, or multiplex assay.

In another preferred embodiment, the target nucleic acid comprises a single-stranded nucleic acid, a double-stranded nucleic acid.

In a ninth aspect of the invention, there is provided a detection system for detecting a target nucleic acid molecule, the system comprising:

The CAS12 enzyme variant or the functional derivative thereof according to the first aspect of the present invention or the second aspect of the present invention;

a guide RNA that directs specific binding of the CAS12 enzyme variant or functional derivative thereof to a target nucleic acid molecule, and a nucleic acid probe.

In another preferred embodiment, the nucleic acid probe is a DNA nucleic acid probe or an RNA nucleic acid probe.

In a tenth aspect of the present invention, there is provided a detection system for selectively detecting a target nucleic acid molecule, the system comprising:

two or more Cas12 proteins or functional derivatives thereof that differ significantly in the cleavage specificity for RNA and DNA;

a guide RNA that directs specific binding of a Cas12 protein or functional derivative thereof to a target nucleic acid molecule, and

A nucleic acid probe;

wherein at least one of the Cas12 proteins or functional derivatives thereof is a Cas12 enzyme variant or functional derivative thereof according to the first aspect of the invention or the second aspect of the invention.

In another preferred embodiment, at least one Cas12 protein that has a significant difference in cleavage specificity for RNA and DNA is any one of the engineered Cas12 enzyme variants described above or a functional derivative thereof. The target nucleic acid molecule can be target RNA or target DNA.

In another preferred embodiment, 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 practical needs.

In another preferred example, the two or more Cas12 proteins or functional derivatives thereof may be two, three, four, or more.

In another preferred embodiment, the detection system comprises a nucleic acid detection composition comprising a Cas12 protein or a functional derivative thereof, a gRNA comprising a region that binds to the Cas12 protein or a functional derivative thereof and a guide sequence that hybridizes to a target sequence on a target nucleic acid, and a nucleic acid probe, detecting a detectable signal generated by cleavage of the nucleic acid probe by the Cas12 protein or a functional derivative thereof, thereby detecting the target nucleic acid.

In another preferred embodiment, the nucleic acid detection composition comprises one or more CAS12 enzyme variants or functional derivatives thereof as described in the first aspect of the invention or the second aspect of the invention, a gRNA that can bind to one or more CAS12 enzyme variants as described in the first aspect of the invention or the second aspect of the invention and that hybridizes to a target sequence on a target nucleic acid, and a nucleic acid probe. In another preferred embodiment, the nucleic acid detecting composition comprises either one or both of the first nucleic acid detecting composition and the second nucleic acid detecting composition:

The first nucleic acid detection composition comprises a first Cas12 protein, a first gRNA that binds to Cas12 enzyme variants and hybridizes to a target sequence on a target nucleic acid, and a first nucleic acid probe;

The second nucleic acid detection composition includes a second Cas12 protein, a second gRNA that binds to Cas12 enzyme variants and hybridizes to a target sequence on a target nucleic acid, and a second nucleic acid probe.

In another preferred embodiment, the first Cas12 protein is a Cas12 enzyme variant having the sequence shown in SEQ ID No.9 (AaCas b-dHax).

In another preferred embodiment, the second Cas12 protein is a second Cas12 enzyme variant having the sequence shown in SEQ ID No.10 (AaCas 12 2b+5mpl).

In another preferred embodiment, the first CAS12 enzyme variant specifically cleaves the first nucleic acid probe, thereby generating a first detectable signal;

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

In another preferred embodiment, the nucleotides of the first nucleic acid probe consist of polyT or polyA

In another preferred embodiment, the nucleotides of the second nucleic acid probe consist of polyT or polyA.

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

In another preferred embodiment, the nucleic acid detection composition further comprises a third Cas12 protein, a third gRNA that binds to the third Cas12 protein and hybridizes to a third target sequence on the target nucleic acid, and a third nucleic acid probe.

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

In another preferred embodiment, the third Cas12 protein specifically cleaves the third nucleic acid probe, thereby generating a third detectable signal.

In another preferred embodiment, the nucleotide of the third nucleic acid probe consists of polyA consisting of 7 consecutive A bases.

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

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

Preferably, both ends of the nucleic acid probe are provided with a fluorescent group and a quenching group, respectively, which can exhibit a detectable fluorescent signal when the nucleic acid probe is cleaved. The fluorescent group is one or more selected from FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED460, and the quenching group is one or more selected from BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

In another preferred example, the first nucleic acid probe and the second nucleic acid probe are respectively provided with a first fluorescent group, a first quenching group, a second fluorescent group and a second quenching group at two ends, wherein the first fluorescent group and the second fluorescent group can be identical or different fluorescent groups, and the first quenching group and the second quenching group can be identical or different quenching groups.

In another preferred example, the first nucleic acid probe, the second nucleic acid probe and the third nucleic acid probe are respectively provided with a first fluorescent group and a first quenching group at both ends, the second fluorescent group, the second quenching group, the third fluorescent group and the third quenching group, the first fluorescent group, the second fluorescent group and the third fluorescent group can be the same or different fluorescent groups, and the first quenching group, the second quenching group and the third quenching group can be the same or different quenching groups.

In another preferred embodiment, the 5 'end and the 3' end of the nucleic acid probe are respectively provided with different reporter groups, or the 5 'end and the 3' end of the nucleic acid probe are respectively provided with different labeling molecules.

In another preferred embodiment, the guide RNA is an RNA that directs Cas protein to specifically bind to the target DNA.

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

In another preferred embodiment, the target nucleic acid is derived from a sample of a virus, bacteria, microorganism, soil, water source, human, animal, plant, etc., and preferably the target nucleic acid is a product enriched or amplified by a method such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.

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 SNP site or a nucleic acid that differs from a control, preferably the virus is a plant virus or an animal virus, e.g., papilloma virus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus, preferably the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV.

In an eleventh aspect of the invention, there is provided a kit for detecting a target nucleic acid in a sample, the kit comprising a nucleic acid detection composition comprising a Cas protein, a gRNA, and a nucleic acid probe, the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid;

The nucleic acid detection composition is selected from one or more CAS12 enzyme variants as described in the first aspect of the invention or the second aspect of the invention, can bind to one or more CAS12 enzyme variants as described in the first aspect of the invention or the second aspect of the invention and gRNA hybridized to a target sequence on a target nucleic acid, and a nucleic acid probe.

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 shows predicting a section of an engineering editable.

FIG. 2 shows the RNA viability test results of the mutants. Wherein Aacas b-PTC is the result of wild type AaCas b protein with target sequence as substrate, aacas b-NTC is the result of wild type AaCas b protein with negative sample as substrate

FIG. 3 shows the deletion verification of the trans-RNase activity of AaCas b-SSoSSB.

FIG. 4 shows the Rnase viability boost verification of AaCas b+5MPL, aaCas12b+dHax3.

FIG. 5 shows that AaCas b-dHax alters the base bias validation of DNA.

FIGS. 6A-6G show different DNA binding protein domains.

Detailed Description

The inventor of the present invention has studied extensively and intensively, and found for the first time that through protein mutational mutation technology, the specificity of RuvC recognition and cleavage of DNA substrate in Cas12b is modified, specifically recognizes DNA substrate or specifically recognizes RNA substrate, and in practical application, solves the problem of insufficient discrimination of DNA/RNA substrate recognition. The RNA probe may be used as a reporter substrate in a system, or the DNA and RNA probes may be used simultaneously in one system, and two or more targets may be reported in parallel by guiding with two specific sgRNAs, respectively. And further, the reaction efficiency of the Cas12b on the RNA substrate after the trans-cleavage activity is opened is improved, and the reporting efficiency of the Cas12b using the RNA probe is improved. The present invention has been completed on the basis of this finding.

Terminology

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

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.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of, or" consisting of.

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).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

"Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity 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%.

As used herein, the "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (Clusteredregularly interspaced short palindromic repeats) derived from the immune system of a microorganism.

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

As used herein, "guide RNA," "sgRNA," and "gRNA" are used interchangeably herein to refer to an RNA capable of forming a complex with a Cas12 protein and a target nucleic acid.

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

As used herein, "complementarity" of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity means 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 50%,60%,70%,80%,90% and 100% complementary for about 5,6,7, 8, 9, 10 of 10, respectively). "fully complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of any one of at least about 70%,75%,80%,85%,90%,95%,97%,98%,99% or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions. For a single base or a single nucleotide, pairing of A with T or U, C with G or I is referred to as complementary or matching, and vice versa, according to Watson-Crick base pairing rules, while other base pairing is referred to as non-complementary or non-matching. "complementary" in the present application includes the cases of "complete complementary" and "substantially complementary", unless otherwise indicated. Two nucleic acid sequences are said to be "complementary" whenever they can form a stable hybrid duplex by Walson-Crick base pairing, and the process by which they form a stable hybrid duplex is referred to as "complementary hybridization".

As used herein, the term "wild-type" has a meaning commonly understood by those skilled in the art, meaning a typical form of an organism, strain, gene or trait that distinguishes it from mutants or variants when it is present in the nature. It can be isolated from resources in nature and is not deliberately modified.

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably to refer to human participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant 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 which naturally occurs.

Cas proteins

As used herein, the term "Cas12" or "Cas12 protein" or "Cas12 enzyme" includes Cas12a (also referred to as Cpf 1), cas12b, cas12c, cas12d, cas12e, cas12h, cas12i, cas12g, and the like. In some embodiments, the Cas12 protein is a Cas12b protein, with Cas12b protein being used in its broadest sense and includes the parent or reference Cas12b protein (e.g., aaCas b with amino acid sequence SEQ ID No. 6), derivatives or variants thereof, and functional fragments, such as oligonucleotide binding fragments thereof.

The Cas protein of 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 that the Cas protein can recognize the PAM locus and specifically cut the target sequence under the action of gRNA.

Cas proteins, such as Cas12, as used herein 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 retains, at least in part, the activity of the protein. Variants can include mutants (which can 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.

Cas12g has strong trans-cleavage activity on both ssDNA and ssRNA, and the structure analysis shows a definite structure of a Zinc Finger domain (Zinc Finger), which is a binding center of many transcription factors and plays a central role in RNA recognition in general cognition. Homologous alignment is performed on Cas12b and Cas12g, a homologous region of the two is found in a RuvC (Nuc) region, a structure homologous to Cas12g1 of an alpha helix matched with RuvC in Cas12b is identified, a potential zinc ion binding domain on the upstream and downstream of the alpha helix is deduced, and the region is a domain responsible for stabilizing nucleic acid substrate binding in Cas12 b. In some embodiments, the zinc finger domain has 1 to 2 zinc binding sites selected from one of [ CxxxxC ], [ CxxxxH ], [ CxxxC ], [ HxxxH ], [ CxxC ], [ CxxH ], wherein x represents any natural amino acid.

As interpreted from the crystal structure of Cas12b protein, this region is adjacent to RuvC nucleic acid hydrolysis domain, and by altering the binding properties of this region, the intensity and specificity of nucleic acid recognition, e.g., DNA recognition, RNA recognition, etc., can be affected.

As used herein, a "Cas12 enzyme variant" is preferably a mutant having only trans-recognition activity on DNA and a mutant with increased RNA trans-reactivity obtained by selecting several different single-stranded nucleic acid binding domains, replacing or substituting the alpha helix of RuvC (Nuc) region and the potential zinc ion binding domains upstream and downstream (zinc finger domains) in original Cas12 b.

Further, a novel method of using RNA trans-cleavage activity as a reporter system has been developed by using a mutant having an improved RNA trans-reactivity.

A system was developed in which the Cas12b mutant simultaneously reported two targets in the same system using a mutant having only a trans-recognition activity for DNA and a Cas12b mutant having an improved RNA trans-reactivity.

As used herein, a "functional derivative" of a certain protein includes various variants or functional domains of the protein, which may be referred to as functional derivatives of the protein, as long as the variants or functional domains retain the function of a certain functional domain of the protein, whether enhanced or reduced. For example, for Cas12 proteins, cas12 protein variants or truncations that retain their partial domain function are functional derivatives of Cas12 proteins

Nucleic acid probe

The nucleic acid probe or single-stranded nucleic acid probe of the present invention comprises different reporter groups or marker molecules at both ends, which when in an initial state (i.e. not cleaved) exhibit no reporter signal, and when the single-stranded nucleic acid is cleaved exhibit a detectable signal, i.e. a detectable distinction between after cleavage and before cleavage. In the present invention, if a detectable difference can be detected, it is reflected that the target nucleic acid contains a feature sequence to be detected, or if the detectable difference cannot be detected, it is reflected that the target nucleic acid does not contain a feature sequence to be detected.

In one embodiment, the reporter or marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED460, and the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

Target nucleic acid

As used herein, the term "target nucleic acid", "target nucleic acid" or "target nucleic acid molecule" refers to a 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.

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 SNP site or a nucleic acid that differs from a control, preferably the virus is a plant virus or an animal virus, e.g., papilloma virus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus, preferably the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV.

In some embodiments, the target nucleic acid is derived from a cell, e.g., from a cell lysate.

In some embodiments, the measurement of the detectable signal may be quantitative, and in other embodiments, the measurement of the detectable signal may be qualitative.

Variants

As used herein, "variant" or "mutant" is interpreted as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains the requisite properties. Typical variants of a polynucleotide differ from the nucleic acid sequence of another reference polynucleotide. Variations in the variant nucleic acid sequence may or may not alter 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 differs in amino acid sequence from another reference polypeptide. In general, the differences are limited such that the sequences of the reference polypeptides and variants are very similar overall and identical in many regions. The amino acid sequences of the variant and reference polypeptides may differ by any combination of one or more substitutions, additions, deletions. The amino acid residues that are replaced or inserted may or may not be those encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring (such as allelic variants), or may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of skill in the art.

The 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 retain the function or activity of the CAS12 enzyme variants of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide having one or several conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound such as a compound which extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (fusion protein formed by fusion with a tag sequence such as a leader sequence, a secretory sequence or 6 His). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides in which up to 3, preferably up to 2, more preferably up to 1 amino acid is replaced by an amino acid of similar or similar nature, as compared to the amino acid sequence of the invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table a conservative variant polypeptide amino acid substitution table

The invention also provides analogs of the CAS12 enzyme variants of the invention. These analogs may differ from the polypeptides of the invention by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

In addition, the CAS12 enzyme variants of the present invention may also be modified. Modified (typically without altering the primary structure) forms include chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.

The term "polynucleotide encoding a variant CAS12 enzyme of the present invention" may include polynucleotides encoding variants of the CAS12 enzymes of the present invention, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as the invention or variants of the CAS12 enzyme. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution pattern of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the CAS12 enzyme variant it encodes.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%.

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

The full-length polynucleotide sequence of the present invention can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Expression vector

The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors of the invention or the CAS12 enzyme variant coding sequences of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant CAS12 enzyme variants by conventional recombinant DNA techniques. Generally, there are the following steps:

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

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the polynucleotide sequence encoding the CAS12 enzyme variant may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the CAS12 enzyme variant encoding DNA sequences of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E.coli, the lambda phage PL promoter, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses and some other known promoters which control gene expression in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits 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 E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, streptomyces, salmonella typhimurium bacterial cells, fungal cells such as yeast, plant cells (e.g.ginseng cells).

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 elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene. Examples include the SV40 enhancer 100 to 270 base pairs on the late side of the origin of replication, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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.

Application of

The application also provides a detection system for detecting a target nucleic acid molecule, the system comprising any one or more of the CAS12 enzyme variants or functional derivatives thereof described above, a guide RNA that directs CAS12 protein or functional derivative thereof to specifically bind to the target nucleic acid molecule, and a nucleic acid probe. The target nucleic acid molecule can be target RNA or target DNA. In some embodiments, the nucleic acid probe is an RNA probe, and the specific composition and length of the RNA probe may be designed according to actual needs.

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 CAS12 enzyme variants or functional derivatives thereof, a guide RNA, and a target nucleic acid molecule described above, and measuring a detectable signal generated by cleavage of a nucleic acid probe by the CAS12 enzyme variant or functional derivative thereof, thereby detecting the target nucleic acid molecule. The target nucleic acid molecule can be target RNA or target DNA.

The application also provides a detection system for selectively detecting a target nucleic acid molecule, the system comprising more than two Cas12 proteins or functional derivatives thereof that differ significantly in cleavage specificity for RNA and DNA, a guide RNA that guides Cas12 proteins or functional derivatives thereof to specifically bind to the target nucleic acid molecule, and a nucleic acid probe. Wherein the two or more Cas12 proteins or functional derivatives thereof may be two, three, four, or more.

In some embodiments, at least one Cas12 protein or functional derivative thereof that has a significant difference in cleavage specificity for RNA and DNA is any one of the Cas12 enzyme variants described above or functional derivatives thereof. The target nucleic acid molecule can be target RNA or target DNA.

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 may be designed according to actual needs.

The main advantages of the invention include

(1) The reformable region of Cas12b identified by the present inventors can be modified by substituting or inserting different single-chain binding proteins, and combining changes, so that the substrate identification of AaCas b enzyme is extended, the original DNAse activity of AaCas b enzyme guided by gRNA is improved, and the range of DNA cleavage from original DNA cleavage to efficient DNA cleavage is expanded.

(2) The muteins of the present invention may be used in vitro assay applications. In addition, the mutated enzyme provided by the invention can specifically identify the DNA probe without cutting RNA substrate after being activated, and can be used as a reporting system independent of the former enzyme after being activated.

The invention is further illustrated below in conjunction with specific embodiments. 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 procedures, which do not address the detailed conditions in the examples below, are generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

Example 1 sequence alignment and mutation region determination of Cas12b and Cas12g

By using the CLUSTAL 2.1 Multiple Sequence Alignments tool, homology alignment was performed on Cas12b and Cas12g, and the similarity of both in RuvC (Nuv) region was found to be relatively high. The structure of alpha helix matched with RuvC in Cas12b, which is homologous to Cas12g1, and the potential sites P916 and L941 (CxxxxC, cxxxxH, cxxxC, hxxxH, cxxC or CxxH) which can form stronger zinc binding on the upstream and downstream of the alpha helix are identified, and the analysis of crystal structure of the combined Cas12b, the existence of gate points which limit the stable entry of larger structural ribose is presumed to be F897 and F964 on the upstream and downstream, and the structures are hidden in the structural domain of 882-992 RuvC.

As shown in FIG. 1, the predicted engineering editable segments are P916-P945.

Example 2 structural mutation of the alpha helix upstream and downstream of the goan amino acid of cas12b and protein acquisition

For the Cas12b predicted engineered editable segments, predicted features are DNA recognition and capture, selecting several structures of nucleic acid binding proteins, including single-chain binding proteins in prokaryotes to mutate wild type AaCas b in the following 9 ways. The results of the post-mutation grouping with ClustalW alignment, the mutated segments are shown below:

substitution mutation alignment of DNA binding protein domains

Alignment of DNA and RNA binding protein domain insertion mutations

(1) The 919-947 region of the amino acid sequence of wild-type AaCas b (the amino acid sequence of this region is CAREQNPEPFPWWLNKFVAEHKLDGCPLR, SEQ ID NO. 1),

A segment replaced with TthSSB protein (the amino acid sequence of the segment is

TAVARLGLAVNERRQGAEERTHFVEVQAWRDLAEWAAELRKGDGLFVIGRLVNDSWTSSSGERRFQTRVEALRLERPTR, SEQ ID No. 2), the resulting protein is designated AaCas b-TthSSB, and the amino acid sequence of the protein is shown as SEQ ID No. 7. Wherein the underlined section indicates the replaced segment.

(2) The 919-947 region of the amino acid sequence of wild-type AaCas b was replaced by a region of SsoSSB protein:

TVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIA, SEQ ID No. 3), the protein obtained is AaCas b-SSoSSB, and the amino acid sequence of the protein is shown as SEQ ID No. 6. Wherein the underlined section indicates the replaced segment.

(3) The 916-944 segment of the amino acid sequence of the wild-type AaCas b is replaced by an effector monomer of the TALE protein (the amino acid sequence of the segment is GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN, SEQ ID NO. 5), and the obtained protein is AaCas b-dHax, and the amino acid sequence of the protein is shown as SEQ ID NO. 15. Wherein the underlined section indicates the replaced segment.

(4) A segment of 5MPL protein was inserted after position 945 (between positions 945 and 946) of the amino acid sequence of wild-type AaCas b (the amino acid sequence of this segment is:

GGGSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRGGGSP,SEQ ID NO.4), The obtained protein is called AaCas b+5MPL, and the amino acid sequence is shown as SEQ ID NO. 10. Wherein the underlined section indicates the inserted section.

(5) After position 945 (between positions 945 and 946) of the amino acid sequence of wild-type AaCas b, a 3x TALE effector monomer was inserted (the amino acid sequence of this segment is:

GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN, SEQ ID No. 5), the resulting protein is designated AaCas12b+ dHax3, and the amino acid sequence of the protein is shown as SEQ ID No. 9. Wherein the underlined section indicates the inserted section.

Designing corresponding vectors according to the amino acid sequences of the 5 mutants, transferring the vectors into a pET28 expression vector, performing induced expression in an escherichia coli strain BL21, and obtaining 5 corresponding mutant proteins through protein purification methods such as affinity chromatography, ion exchange chromatography and the like.

EXAMPLE 3 preliminary determination of mutant Activity

The final system concentration for the viability assay was 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul Cas12b protein, 500nM probe, 42℃as shown in the table, and 1h of reaction. HEX channel fluorescence was collected on a macrostone SLAN 96 instrument, and the final results were displayed with DNA viability assay results selected using the initial fluorescence values and RNA viability assay results selected using the final fluorescence values.

Table B different protein mutant DNA activity determination system and related sequence

Component name	Concentration of use	Final concentration	1rxn/ul
				ddH2O	/	/	4
rCutSmart	10X	1X	2
				Rnase inhibitor	20U/μL	0.5U/μL	0.5
Cas12b protein mutants	600ng/ul	60ng/ul	2
				sgRNA2	300ng/ul	7.5ng/ul	0.5
Probe1	5μM	500nM	2
				Sample of 2	2E11copies/ul	1E12copies	5

1. Sequence information of the probe

DNA or RNA probes	Sequence(s)
		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

2. SgRNA and template sequence information

As shown in FIG. 2, it can be seen that the mutants have the following changes

AaCas12b-TthSSB loses the trans-cleavage activity of DNA and RNA after replacing the original binding region;

AaCas12b-SSoSSB replaces the original binding region, loses the trans-cutting activity of RNA, retains the trans-cutting activity of DNA, changes the clipping preference of the original trans-cutting activity of DNA, and becomes an incompatible A base;

AaCas12b-dHax changes the base preference after replacing the original binding region, and the trans-cutting activity of DNA becomes more preferential to T bases and does not have affinity to A and C bases;

AaCas12b+5MPL retains the trans-cleavage activity of DNA, and thus obtains higher RNA trans-cleavage activity;

AaCas12b+ dHax3, on the basis that the trans-cutting activity of DNA is reserved, the higher RNA trans-cutting activity is obtained;

example 4 Trans RNase Activity deficiency verification by AaCas12b-SSoSSB

AaCas12bSSoSSB verifies the cleavage system;

The final system concentration for the viability assay, 1xrCutSmart buffer,7.5ng/ul sgRNA,60ng/ul Cas12b protein, 500nM RNA probe, shown in Table C, 42℃for 1h. Ion MnCl ₂、CaCl₂、CoCl₂, SPERMIDINE and 42 ℃ are added in addition to the above, and the reaction is carried out for 1h. HEX channel fluorescence was collected on a macrostone SLAN 96 instrument and the final results were counted with the maximum increase in fluorescence per unit time.

Table C AaCas12b-SSoSSB RNA cleavage Activity verification System under different cofactors

1. Detailed information of the probe

	Sequence(s)
		rA—RNA—BHQ1	5’FAM—rArArArArArArA—3’BHQ1

2. SgRNA and template sequence information

As can be seen from FIG. 3 above, aaCas b-SSoSSB is not capable of activating trans-RNase activity under various additive conditions.

Example 5 Rnase Activity elevation verification of AaCas12b+5MPL AaCas12b+dHax3

AaCas12 MPL, aaCas12b+dHax3, verify the cleavage system;

The final system concentration for the viability assay, 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul Cas12b protein, 500nM RNA probe, as shown in Table D, 42℃for 1h. HEX channel fluorescence was collected on a macrostone SLAN 96 instrument and the final results were counted with the maximum increase in fluorescence per unit time.

Table D AaCas12b+5MPL,AaCas12b+dHax3 RNA cleavage viability validation system

Component name	Concentration of use	Final concentration	1rxn/ul
				ddH2O	/	/	To 20ul
rCutSmart	10X	1X	2
				Rnase inhibitor	20U/μL	0.5U/μL	0.5
Cab12 protein mutant	600ng/ul	60ng/ul	2
				sgRNA2	300ng/ul	7.5ng/ul	0.5
Probe1	5μM	500nM	2
				Sample of 2	2E11copies/ul	1E12copies	5

1. Detailed information of the probe

	Sequence(s)
		rA—RNA—BHQ1	5’FAM—rArArArArArArA—3’BHQ1

2. SgRNA and template sequence information

As shown in FIG. 4, 1,2,3 are AaCas b+5MPL, aaCas12b+dHax3, and wild type AaCas b is a comparison of trans and Rnase activities.

Example 6 AaCas12b-dHax altered base bias validation of DNA

The three proteins AaCas b, aaCas12b-dHax and AaCas12b-SSoSSB are added into a cleavage reaction system, and the final reagent concentration is 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul Cas12b protein, a template and 500nM are reacted for 1h by using DNA probes with two different positions of thio and different fluorophores at 42 ℃. HEX channel fluorescence was collected on a macrostone SLAN 96 instrument and the final results were counted with the maximum increase in fluorescence per unit time.

Table E AaCas12b-dHax cleavage preference verification System

Component name	Concentration of use	Final concentration	1rxn/ul
				ddH2O	/	/	To 20ul
rCutSmart	10X	1X	2
				Rnase inhibitor	20U/μL	0.5U/μL	0.5
Cab12 protein mutant	600ng/ul	60ng/ul	2
				sgRNA2	300ng/ul	7.5ng/ul	0.5
Probe1	5μM	500nM	2
				Sample of 2	2E11copies/ul	1E12copies	5

1. Detailed information of the probe

Probe with a probe tip	Sequence(s)
		HEX-DNA-probe	5‘HEX-TT*T*T*T*T*T-3’BHQ1
FAM-DNA-probe	5‘FAM-C*C*C*C*C*CC-3’BHQ1

2. SgRNA and template sequence information

As shown in FIG. 5, the ratio of viability to dT and dC was changed after AaCas b-dHax replaced the original nucleic acid domain.

Example 7 detection Using RNA probes Using AaCas b+5MPL, aaCas12b+ dHax3, cas12i

AaCas12b+5MPL at 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul AaCas12b+5MPL protein, novel coronavirus sample, 500nM RNA probe, 42℃for 1h.

AaCas12b+ dHax3 at 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul AaCas12b+ dHax3 protein, novel coronavirus sample, 500nM RNA probe, 42℃for 1h.

Cas12i at 1x rCutSmart buffer,7.5ng/ul sgRNA,60ng/ul Cas12i protein, novel coronavirus sample, 500nM RNA probe, 42℃for 1h.

The results show that all three proteins AaCas b+5MPL, aaCas12b+ dHax3 and Cas12i can successfully detect the novel coronavirus positive sample.

Example 8 Dual detection of DNA probes in one System Using Cas12b-dHax, and Cas12i

Cas12b-dHax and Cas12i, with two DNA probes of different positions thio and different fluorophores, are added into the same system, the Cas12b-dHax DNA probe is FAM fluorescence, and the Cas12i is HEX fluorescence.

Table F DNA probe dual detection system

Component name	Concentration of use	Final concentration	1rxn/ul
				ddH2O	/	/	To 20ul
rCutSmart	10X	1X	2
				Rnase inhibitor	20U/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
HEX-DNA-probe1	5μM	500nM	2
				FAM-DNA-probe1	5μM	500nM	2
Novel coronavirus sample			2.5
				HPV samples			2.5

1. Probe information

	Sequence(s)
		HEX-DNA-probe	5‘HEX-TT*T*T*T*T*T-3’BHQ1
FAM-DNA-probe	5‘FAM-C*C*C*C*C*CC-3’BHQ1

The result shows that the novel coronavirus sample and the HPV sample can collect corresponding fluorescent signals in different fluorescent channels in the same reaction system, and positive samples can be successfully detected.

Example 9 Dual detection of RNA probes in one System Using Cas12b-dHax, and Cas12i

Cas12b-dHax and Cas12i, with RNA probes of two different positions thio and different fluorophores, are added into the same system, the Cas12b-dHax RNA probe is FAM-RNA-probe, and the Cas12i is HEX-RNA-probe.

Table G RNA probe dual detection system

Component name	Concentration of use	Final concentration	1rxn/ul
				ddH2O	/	/	To 20ul
rCutSmart	10X	1X	2
				Rnase inhibitor	20U/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
HEX-RNA-probe1	5μM	500nM	2
				FAM-RNA-probe1	5μM	500nM	2
Novel coronavirus sample			2.5
				HPV samples			2.5

1. Probe information

	Sequence(s)
		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

The result shows that the novel coronavirus sample and the HPV sample can collect corresponding fluorescent signals in different fluorescent channels in the same reaction system, and positive samples can be successfully detected.

Discussion:

The present application utilizes RuvC and NuC regions in Cas12b to improve recognition of the specificity of cleaving DNA substrates. Different Cab12 mutants are obtained by replacing or inserting different single-stranded binding proteins or combinations thereof, e.g. different nucleic acid binding proteins (segments of TthSSB protein, segments of SsoSSB protein, segments of 5MPL protein, TALE effector concatamers). The Cab12 mutant expands the substrate recognition of the Cas12b enzyme, can specifically recognize a DNA substrate or an RNA substrate (namely a single-stranded nucleic acid probe), and can be used for performance modification of other Cas12 enzymes containing NuC regions. Through the transformation, the double or multiple detection of the mutant of the Cas12b is realized in the same system.

The sequence of the invention:

The 919-947 region of the amino acid sequence of wild-type AaCas b of SEQ ID NO.1

CAREQNPEPFPWWLNKFVAEHKLDGCPLR

Segment of SEQ ID NO.2 TthSSB protein

TAVARLGLAVNERRQGAEERTHFVEVQAWRDLAEWAAELRKGDGLFVIGRLVNDSWTSSSGERRFQTRVEALRLERPTR

Segment of SEQ ID NO.3 SsoSSB protein

TVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIA

SEQ ID NO. 45 segment of the MPL protein

GGGSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRGGGSP

SEQ ID NO. 53 xTALE effector monomer

GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN

SEQ ID NO:6>WT AapCas12b

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO.7 AaCas12b-TthSSB

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARTAVARLGLAVNERRQGAEERTHFVEVQAWRDLAEWAAELRKGDGLFVIGRLVNDSWTSSSGERRFQTRVEALRLERPTRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO.8 AaCas12b-SSoSSB

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARTVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIAADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO.9 AaCas12b-dHax

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO.10 AaCas12b+5MPL

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPGGGSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRGGGSPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO.11 AaCas12b+dHax3

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI*

SEQ ID NO:20>WT Cas12g1

MKIEEGKGHHHHHHMAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQWAIQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPAFTNELRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNYEEWKKEKQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKHSVLCNQFNEFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEETLRGKNGGRLPHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRKPVFRYPSVKRHSIAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGETEISSVHLHFVGTRPRIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLLETFPGNAEQELRLLAVDLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRDASSKQPRPGLSRDHVGRHLQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRGLTVHTARMIRDWARLNARQIIQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHGRIRRKVTEKAVERGMRVVTVPYLASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQVDENAARVLGRVFWGEIELPTAIP

SEQ ID NO:21 Cas12b-ggg

MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARGGGGGGPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI

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 (10)

1. A CAS12 enzyme variant or a functional derivative thereof, 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, characterized in that the segment replacement or insertion of the RuvC and/or Nuc active center of the wild-type CAS12 enzyme that recognizes and cuts the DNA substrate is a domain replacement or insertion of part or all of the region in positions 919-947, wherein the amino acid sequence of positions 919-947 is as shown in SEQ ID NO: 1. 3. The CAS12 enzyme variant or a functional derivative thereof according to claim 1, 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 according to claim 3, characterized in that the CAS12 enzyme variant or a functional derivative thereof 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, 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 vector, characterized in that the vector contains the polynucleotide according to claim 5. 7. A host cell, 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, 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, comprising: 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.