A miniaturized CRISPR / Cas12 hacker gene cutting system

Abstract

This invention relates to the field of gene editing, and in particular to a very small CRISPR / Cas12hacker gene cutting system. The CRISPR / Cas12 gene cutting system includes a Cas12hacker nuclease or a polynucleotide encoding the Cas12hacker nuclease, and also includes guide RNA or a polynucleotide encoding the guide RNA. By binding to the host factor thioredoxin TrxA, the CRISPR-Cas12 gene cutting system enhances the efficiency of the Cas12hacker nuclease in precisely cutting double-stranded DNA, thereby achieving genomic DNA double-strand breaks and highly efficient in vitro DNA double-strand cutting.

Description

Technical Field

[0001] This invention relates to the field of gene editing, specifically to a very small gene cutting and gene editing system, and particularly to a gene cutting method that uses a CRISPR / Cas12 hacker system in synergy with the host factor thioredoxin. Background Technology

[0002] The antagonistic co-evolution of bacteria and exogenous mobile genetic elements (MGEs) exhibits a dynamic array of defense-antagonistic strategies. Among these, clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes (Cas) function as an adaptive immune system in many archaea and bacteria, representing a key bacterial defense strategy that enables the host to resist MGEs. Bacteria utilize CRISPR arrays as molecular archives to capture and record target sequences from invading genomes, allowing Cas proteins to precisely target and cleave these MGEs. Conversely, MGEs, particularly bacteriophages, have also evolved sophisticated countermeasures to circumvent CRISPR / Cas-mediated defenses. These include producing anti-CRISPR (Acr) proteins and RNAs to inhibit Cas effector function or disrupt CRISPR complex assembly, posing a significant challenge to bacterial immunity. In response, bacteria have developed novel antagonistic strategies, such as the IF-type CRISPR system, which can trigger abortion-like infection mechanisms to eliminate Acr-containing elements. In addition, some type II-C CRISPR systems utilize pro-CRISPR proteins to enhance their targeting efficiency for MGEs.

[0003] The Cas12 family is characterized by its evolutionary versatility, exhibiting broad structural and functional diversity and serving as a prime example of the co-evolutionary complexity of host-phage interactions. To date, 15 distinct V-type systems (VA to O) have been identified. The structural diversity of the major domains (WED, REC, and RuvC) of the Cas12 family, ranging in size from 400 to 1500 amino acids, highlights the adaptive potential of these systems. For example, Cas12k cooperates with tn7-like transposases for RNA-guided site-specific transposition, while Cas12f employs a unique homodimeric structure crucial for DNA targeting. Conversely, Cas12m deviates from conventional DNA cleavage patterns, opting instead for gene silencing to combat the threat of MGEs.

[0004] While various anti-CRISPR mechanisms have been discovered in bacteriophages to disrupt these immune systems, research on the role of bacterial host factors in enhancing CRISPR / Cas function is relatively limited. Therefore, exploring and studying novel CRISPR / Cas systems and investigating novel evolutionary mechanisms for bacterial immune defense is of great importance. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an extremely small CRISPR / Cas12hacker gene cutting system to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides the use of Cas12 hacker nuclease or polynucleotide encoding said Cas12hacker nuclease in the preparation of CRISPR / Cas12 gene cutting systems, gene editing systems or in gene cutting or gene editing.

[0007] The present invention also provides a CRISPR / Cas12 gene cutting or gene editing system, the CRISPR / Cas12 gene cutting or editing system comprising:

[0008] 1) Cas12 hacker nuclease or a polynucleotide encoding the Cas12 hacker nuclease;

[0009] 2) Guide RNA or polynucleotide encoding the guide RNA.

[0010] The present invention also provides a recombinant expression vector comprising a nucleotide sequence encoding a Cas12 hacker nuclease, a nucleotide sequence encoding a guide RNA, and / or a nucleotide sequence encoding a thioredoxin.

[0011] The present invention also provides a gene cutting method, comprising mixing the gene cutting or gene editing system with a target sequence or cells containing the target sequence to perform gene cutting.

[0012] The present invention also provides a cell, which is obtained by cutting or editing using the gene cutting or gene editing system described above.

[0013] As described above, the miniature CRISPR / Cas12 hacker gene cutting system of the present invention has the following beneficial effects: the miniature CRISPR-Cas12 hacker system can enhance the efficiency of Cas12 hacker nuclease in precisely cutting double-stranded DNA by binding to the host factor thioredoxin TrxA, so as to achieve genomic DNA double-strand breaks and efficient cutting of DNA double strands in vitro. Attached Figure Description

[0014] Figure 1 The diagram shows the identification process and results of the neighboring motif (PAM) in the anterior interstitial region of the Cas12 hacker.

[0015] Figure 2 This is a diagram showing the results of cutting the substrate DNA using the Cas12 hacker nuclease.

[0016] Figure 3 This diagram shows the results of cutting a 56bp fluorescently labeled DNA substrate using the Cas12 hacker nuclease.

[0017] Figure 4 This presents the results of an investigation into the biochemical reaction condition preferences of the Cas12 hacker. Figure 4 Figure a shows the temperature preference of the Cas12 hacker nuclease. The optimal temperature for Cas12 hacker to cut DNA is between 30 and 40 degrees Celsius. Figure 4 b-4d are graphs showing the Cas12hacker nuclease's preference for different types of divalent cations; Figure 4 Figures e and 4f show the results of the Cas12 hacker nuclease's preference for NaCl concentration.

[0018] Figure 5 The electron microscopy sample preparation and collection process results for the Cas12 hacker-sgRNA-DNA complex. Among them, Figure 5 a and 5b are gel images and molecular sieve peak diagrams of the Cas12 hacker-sgRNA-DNA complex preparation process; Figure 5 c is a structural diagram of the Cas12hacker-TrxA-sgRNA-DNA complex.

[0019] Figure 6 A schematic diagram and results of the CRISPR-Cas12 hacker system targeting bacterial genome cleavage in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Figure 6 a represents the methods and processes of genome cutting. Figure 6 b shows the results of genome cleavage in E. coli using Cas12hacker nuclease. Figure 6 c shows the results of genome cleavage in Klebsiella pneumoniae using the Cas12 hacker nuclease. Figure 6 Figure d shows the results of genome cleavage in Acinetobacter baumannii using the Cas12 hacker nuclease.

[0020] Figure 7A schematic diagram and results of T4 phage infection of Escherichia coli containing the CRISPR-Cas12 hacker system. Figure 7 a represents the process by which bacteriophages infect Escherichia coli. Figure 7 b is a graph showing the results of the CRISPR-Cas12 hacker system resisting infection by T4 phage.

[0021] Figure 8 Analysis of key amino acid sites for the interaction between Cas12 hacker and TrxA. Figure 8 Figure a shows a detailed structural diagram of the key amino acid sites involved in the interaction between the Cas12 hacker and thioredoxin TrxA. Figure 8 b represents a comparison of the effects of the Cas12 hacker mutant on bacterial genome cleavage efficiency. Figure 8 c represents a comparison of the effects of the TrxA mutant on bacterial genome cleavage efficiency.

[0022] Figure 9 Analysis of key amino acid sites for the interaction between the Cas12 hacker and nucleic acids. Figure 9 Figure a shows the structural details of the key amino acid sites where the Cas12 hacker interacts with nucleic acids. Figure 9 b is a diagram showing the results of in vitro cleavage of double-stranded DNA by the Cas12 hacker single mutant. Figure 9 c shows the results of in vitro cleavage of double-stranded DNA by Cas12 hacker triple, quadruple, and heptamericans.

[0023] Figure 10 This is a comparison of the double-stranded DNA cleavage efficiency of the Cas12 hacker wild-type protein and the RuvC domain mutant protein. Figure 10 In the diagram, 'a' represents the structure of the Cas12 hacker RuvC domain. Figure 10 Figure b shows the comparison between the double-strand DNA cleavage activity of the three mutants and the wild-type Cas12 hacker protein.

[0024] Figure 11 The figure shows the effect of thioredoxin TrxA on the in vitro double-stranded DNA cleavage activity of Cas12 hacker. Without TrxA, the in vitro double-stranded DNA cleavage activity of Cas12 hacker is very weak; supplementation with TrxA enhances its cleavage activity. Detailed Implementation

[0025] This invention studies the interaction between existing CRISPR / Cas systems and host factors, discovering a method for host factors to assist CRISPR / Cas systems in genome cleavage. Based on this, this invention provides a novel CRISPR / Cas system and genome cleavage method based on a new miniature CRISPR / Cas12hacker nuclease. Using the CRISPR / Cas system or method of this invention, precise sequence cleavage of target genes can be performed within cells; in in vitro cleavage experiments, the target gene sequence can be precisely cut.

[0026] The present invention first provides the use of Cas12 hacker nuclease or polynucleotide encoding said Cas12 hacker nuclease in the preparation of CRISPR / Cas12 gene cutting system, CRISPR / Cas12 gene editing system or in gene cutting or gene editing.

[0027] The Cas12 hacker nuclease is either wild-type Cas12 hacker nuclease or a Cas12 hacker nuclease mutant.

[0028] In some embodiments of the present invention, the wild-type Cas12 hacker nuclease may be a conventional Cas12hacker nuclease derived from the metagenomics of Cas12 hacker-1 to Cas12 hacker-43.

[0029] The amino acid sequence of the wild-type Cas12 hacker nuclease is shown in any one of SEQ ID NO.1 to 43.

[0030] In some embodiments of the present invention, the Cas12 hacker nuclease mutant is a nuclease mutant with cleavage activity, and the nuclease mutant with cleavage activity is selected from any of the following:

[0031] 1) The Cas12 hacker nuclease mutant has the same amino acid sequence as the wild-type Cas12hacker nuclease shown in SEQ ID NO.1 at the following sites: W44, Y45, W47, L51, W102, F105, L107, I110, I111, V113, L115, C127, I129, and I172. These amino acid residues are key amino acid sites for the interaction between the Cas12 hacker nuclease and TrxA.

[0032] 2) The Cas12 hacker nuclease mutant has the same amino acid sequence as the RuvC domain of the wild-type Cas12 hacker nuclease, or the Cas12 hacker nuclease mutant has the same amino acid sequence as the wild-type Cas12 hacker nuclease as shown in SEQ ID NO.1 at the following sites: D378, E478 and D582.

[0033] 3) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease shown in SEQ ID NO.1. The mutation sites include any three or fewer (e.g., 0, 1, 2, or 3) sites selected from K137, R138, K149, K150, K156, K159, and K162. The aforementioned amino acid residues are key amino acid sites for the interaction between the Cas12 hacker nuclease and nucleic acids. Mutations at four or more sites will affect the cleavage ability of double-stranded DNA.

[0034] In some embodiments of the present invention, the Cas12 hacker nuclease mutant is a nuclease mutant with no cleavage activity or reduced cleavage activity relative to the wild type, and the nuclease mutant is selected from any of the following:

[0035] 1) The Cas12 hacker nuclease mutant refers to the Cas12 hacker nuclease mutant obtained by mutation based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1. The mutation sites are selected from any one or more of the following: W44, Y45, W47, L51, W102, F105, L107, I110, I111, V113, L115, C127, I129, and I172. The aforementioned amino acid residues are key amino acid sites for the interaction between the Cas12 hacker nuclease and TrxA. Preferably, the Cas12 hacker nuclease mutant is obtained by mutation of the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1 in one or more of the following ways: W44A, Y45A, W47A, L51A, W102A, F105A, L107A, I110A, I111A, V113A, L115A, C127A, I129A, I172A, C127S.

[0036] 2) The Cas12 hacker nuclease mutant is obtained by mutation of the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation site is selected from any one or more of the following: D378, E478, and D582. Preferably, the Cas12 hacker nuclease mutant is obtained by mutation of any one of the following: D378A, E478A, or D582A, based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1.

[0037] 3) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease shown in SEQ ID NO.1. The mutation sites include any one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) sites selected from K137, R138, K149, K150, K156, K159, and K162. The aforementioned amino acid residues are key amino acid sites for the interaction between the Cas12 hacker nuclease and nucleic acids. Mutations at four or more sites will severely affect the cleavage ability of double-stranded DNA. Preferably, the Cas12 hacker nuclease mutant is obtained by performing any one of the following mutations on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1: single mutation of K137A, R138A, K149A, K150A, K156A, K159A or triple mutation of K149A-K150A-K156A, quadruple mutation of K137A-R138A-K159A-K162A or seven mutation of K137A-R138A-K149A-K150A-K156A-K159A-K162A.

[0038] The aforementioned Cas12 hacker nuclease mutants without cleavage activity, or inactivated Cas12 hacker nuclease mutants, have the following applications: developing Cas12hacker-based single-base editing systems by fusing inactivated Cas12 hacker nuclease mutants with base deaminases; developing Cas12 hacker-based Prime editing systems by fusing inactivated Cas12 hacker nuclease mutants with reverse transcriptases; developing Cas12 hacker-based transcriptional activation systems by fusing inactivated Cas12 hacker nuclease mutants with transcriptional activators; developing Cas12 hacker-based epigenetic modification systems by fusing inactivated Cas12hacker nuclease mutants with nucleic acid epigenetic modification enzymes; and developing Cas12 hacker-based transcriptional repression systems using inactivated Cas12 hacker nuclease mutants, etc.

[0039] In this invention, other amino acid sites of the Cas12 hacker nuclease mutant, besides those mentioned above, are not specifically limited. The Cas12 hacker nuclease mutant can be formed through modification, mutation, DNA shuffling, etc., to give the Cas12 hacker nuclease variant improved desired characteristics, such as function, activity, kinetics, half-life, etc. Modifications may include, for example, the deletion, insertion, or substitution of amino acids, or, for example, the replacement of the "cleavage domain" of the Cas12hacker nuclease with a homologous or heterologous cleavage domain from different nucleases (e.g., the HNH domain of CRISPR-related nucleases); any modification method known in the art for DNA binding and / or DNA-modifying proteins, such as methylation, demethylation, acetylation, etc., can, for example, alter the DNA targeting of the Cas12 hacker nuclease. DNA shuffling refers to exchanging sequence fragments between DNA sequences of Cas12 hacker nucleases from different sources to produce a chimeric DNA sequence encoding a synthetic protein with RNA-directed endonuclease activity. Modifications, mutations, DNA shuffling, etc., can be used alone or in combination.

[0040] Those skilled in the art can choose whether or not a mutant has Cas12 hacker nuclease cleavage activity based on actual needs.

[0041] Specifically, the Cas12 hacker nuclease described in this invention can be:

[0042] (I) Wild-type Cas12 hacker nuclease or a fragment thereof, having RNA-guided nucleic acid binding activity; the amino acid sequence of the Cas12hacker-1 nuclease is shown in SEQ ID NO.1;

[0043] (II) variants that have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence homology with the amino acid sequence of (I) and have RNA-guided nucleic acid binding activity;

[0044] (III) According to (I) or (II), it further includes a nuclear localization signal segment;

[0045] (IV) According to (I) or (II) or (III), the Cas12 hacker nuclease has endonuclease activity.

[0046] Cas12 hacker nuclease variants may have the following specific properties, including but not limited to:

[0047] It has enhanced or reduced ability to bind to the target site, or retains the ability to bind to the target site;

[0048] It has enhanced or reduced ribonuclease and / or nuclease activity, or retains ribonuclease and / or nuclease activity;

[0049] It has deaminase activity, which can act on cytosine, guanine or adenine bases, and then replicate through the deamination site and repair in the cell to produce guanine, thymine and guanine respectively.

[0050] It has the activity of regulating the transcription of target DNA, which can either increase or decrease the transcription of target DNA at specific locations in the target DNA;

[0051] It has altered DNA targeting;

[0052] To increase, decrease, or maintain stability;

[0053] It can cleave the complementary strand of the target DNA, but has a reduced ability to cleave the non-complementary strand of the target DNA;

[0054] It can cleave the non-complementary strand of the target DNA, but has a reduced ability to cleave the complementary strand of the target DNA;

[0055] It has the ability to reduce the cutting of both the complementary and non-complementary strands of the target DNA.

[0056] In some embodiments, the Cas12 hacker nuclease variant has no cleavage activity. In some embodiments, the Cas12hacker nuclease variant has single-strand cleavage activity. In some embodiments, the Cas12 hacker nuclease variant has double-strand cleavage activity.

[0057] Enhanced activity or ability refers to an increase of at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% in activity or ability relative to wild-type Cas12 hacker nuclease.

[0058] Reduced activity and capacity refer to activities or capacities that are less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% relative to wild-type Cas12 hacker nucleases.

[0059] Unless otherwise stated, the terms "Cas12 hacker" and "Cas12 hacker nuclease" include wild-type Cas12 hacker nuclease and all its variants. Those skilled in the art can determine the type of Cas12hacker nuclease variant by conventional means, without being limited to those exemplified above.

[0060] In this invention, the polynucleotide encoding the Cas12 hacker nuclease can be synthesized artificially using existing techniques, such as chemical synthesis, thereby allowing for easy modification in various ways. The modifications can be any method known in the art. In some embodiments, the polynucleotide encoding the Cas12 hacker nuclease contains one or more modifications, allowing for the easy incorporation of numerous modifications, such as enhancing transcriptional activity, altering enzyme activity, improving translational or stability (e.g., increasing resistance to proteolysis or degradation) or specificity, altering solubility, altering delivery, or reducing the innate immune response in host cells. These modifications can be any method known in the art. In some embodiments, the DNA or RNA encoding the Cas12 hacker nuclease introduced into the cell through modification can edit any one or more genomic loci. In some embodiments, the nucleic acid sequence encoding the Cas12 hacker nuclease is a modified nucleic acid, such as codon-optimized. The modifications can be single modifications or combinations of modifications.

[0061] In this invention, the Cas12 hacker nuclease, or the polynucleotide encoding the Cas12 hacker nuclease, is suitable for any biological or in vitro environment, including but not limited to bacteria, archaea, fungi, protozoa, plants, or animals. Accordingly, suitable target cells include, but are not limited to, eukaryotic and prokaryotic cells, such as bacterial cells, archaea cells, fungal cells, protozoan cells, plant cells, or animal cells. The cells can be in vivo or in vitro. In some embodiments, the Cas12 hacker nuclease, or the nucleic acid encoding the Cas12 hacker nuclease, is formulated in liposomes or lipid nanoparticles.

[0062] In this invention, the gene cleavage is either extracellular double-stranded DNA cleavage or intracellular genome cleavage, wherein the genome cleavage is selected from bacterial genome cleavage, viral genome cleavage, etc. In some embodiments of this invention, the gene cleavage is for purposes other than disease diagnosis or treatment.

[0063] The present invention also provides a CRISPR / Cas12 gene cutting system, the CRISPR / Cas12 gene cutting system comprising:

[0064] 1) Cas12 hacker nuclease or a polynucleotide encoding the Cas12 hacker nuclease;

[0065] 2) Guide RNA or polynucleotide encoding the guide RNA.

[0066] The Cas12 hacker nuclease is either the aforementioned wild-type Cas12 hacker nuclease or a Cas12 hacker nuclease mutant.

[0067] The guide RNA (sgRNA) contains a trans-activating CRISPR RNA (tracrRNA) sequence and a crRNA sequence; the crRNA sequence contains a target sequence capable of hybridizing with the target sequence and a tracr pairing sequence.

[0068] The targeting sequence is a nucleotide sequence complementary to the target sequence in the target gene, located at the 3' end of the crRNA sequence. The targeting sequence targets a nucleic acid fragment of 18–22 bp following the PAM sequence. In one embodiment, the targeting sequence is preferably an RNA sequence corresponding to a nucleic acid fragment of 18–22 bp, for example, 20 bp, following the PAM sequence. Preferably, the PAM sequence is 5'-GTN, where N is A, T, C, or G.

[0069] The tracr pairing sequence is ligated to the tracrRNA sequence to form a guide RNA backbone sequence, meaning the sgRNA contains both an RNA backbone sequence and a target sequence. The tracr pairing sequence and the tracrRNA sequence are complementary base-paired, forming a stem-loop structure. This stem-loop structure forms a protein-binding structure that interacts with nucleases.

[0070] In a preferred embodiment of the present invention, the backbone sequence of the guide RNA comprises an optimized and modified backbone sequence of SEQ ID NO. 46 (i.e., SEQ ID NO. 50) of different lengths while still retaining its cleavage activity on the genome. The optimization and modification refer to the sequence obtained by adding, deleting, or replacing some nucleotides at the 5' and / or 3' ends of the backbone sequence shown in SEQ ID NO. 50.

[0071] In some embodiments, the sequence of the guide RNA includes a target sequence as shown in SEQ ID NO.49 or a backbone sequence including nucleotide sequences as shown in any one of SEQ ID NO.50-52.

[0072] In the gene cleavage system, the Cas12 hacker nuclease and sgRNA can form a complex in the host cell that recognizes the PAM sequence on the target gene (e.g., target DNA) sequence. The target sequence of the CRISPR / Cas12 hacker gene cleavage system is a nucleic acid fragment (e.g., a DNA fragment) 20 bp in length following the PAM sequence. In some embodiments, the complex can selectively regulate the transcription of the target DNA in the host cell. The CRISPR / Cas12 hacker gene cleavage system can cut the double strand of the target DNA, causing DNA breaks.

[0073] In some embodiments of the present invention, the gene cutting system further includes thioredoxin or a polynucleotide encoding thioredoxin. Specifically, the CRISPR / Cas12 gene cutting system used for cell delivery contains thioredoxin. The thioredoxin is thioredoxin TrxA.

[0074] In other embodiments of the present invention, if the cell to be cut contains the gene encoding the thioredoxin, then the gene cutting system does not need to include the thioredoxin.

[0075] The thioredoxin TrxA is derived from microorganisms or mammalian cells; the microorganism is, for example, *Escherichia coli*. The thioredoxin is *TrxA* derived from *E. coli*, with the amino acid sequence shown in SEQ ID NO. 47 and the nucleotide sequence shown in SEQ ID NO. 48.

[0076] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNI DQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLA(SEQ ID NO.47);atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactg accgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggcg(SEQ ID NO.48).

[0077] In some embodiments of the present invention, the CRISPR / Cas12 gene cutting system is an extracellular gene cutting system.

[0078] Based on the total volume of the extracellular gene cleavage system, the protein concentration ratio of the Cas12 hacker nuclease to thioredoxin TrxA is 1:(0.5-40), for example 1:(0.5-1), 1:(1-5), 1:(5-10), 1:(10-15), 1:(15-20), 1:(20-25), 1:(25-30), 1:(30-35), 1:(35-40).

[0079] This invention discovers the mechanism by which the CRISPR / Cas12 hacker system collaborates with thioredoxin TrxA to exert nuclease cleavage activity. This mechanism includes key amino acid residues involved in the interaction between the Cas12 hacker nuclease and thioredoxin TrxA, and their binding interface; as well as key amino acid residues involved in the interaction between the Cas12 hacker nuclease and nucleic acids, and their binding interface. The nuclease domains that interact with thioredoxin TrxA and nucleic acids are a thioredoxin binding (TB) domain and a REC domain. The thioredoxin binding domain consists of a pair of parallel β chains.

[0080] The extracellular gene cutting system also includes Mg 2+ or Mn 2+ Based on the total volume of the gene cutting system, the Mg 2+ or Mn 2+ The concentration is 5-20 mM, for example, 5-10 mM, 10-15 mM, 15-20 mM.

[0081] In some embodiments of the present invention, the Mg 2+ or Mn 2+ Provided by MgCl2 or MnCl2.

[0082] In some embodiments of the present invention, the extracellular gene cutting system further includes NaCl.

[0083] When the CRISPR / Cas12 gene cutting system includes Mg 2+ At that time, the concentration of NaCl was 25-100 mM.

[0084] When the CRISPR / Cas12 gene cutting system includes Mn 2+ At that time, the concentration of NaCl was 25-150 mM.

[0085] In some embodiments of the present invention, the extracellular gene cutting system further includes a pH buffer solution. The pH of the pH buffer solution is 7.4 to 7.6. The pH buffer solution can be a conventional pH buffer solution in the art, such as Tris-HCl.

[0086] In some embodiments of the present invention, the CRISPR / Cas12 gene cutting system contains 100 mM NaCl, 10 mM MgCl2, and 10 mM Tris-HCl at pH 7.5, based on the total volume of the gene cutting system.

[0087] In this invention, the concentration of the Cas12 hacker nuclease can be referenced to the concentration of other Cas nucleases used in the art during the reaction. For example, the concentration of the Cas12 hacker nuclease is 300 nM.

[0088] The components of the CRISPR / Cas12 gene cleavage system described in this invention can be delivered via vectors. For example, for polynucleotides, methods that can be used include, but are not limited to, nanoparticles, liposomes, ribonucleoproteins, small RNA conjugates, chimeras, and RNA-fusion protein complexes.

[0089] The present invention also provides a CRISPR / Cas12 gene editing system, wherein the CRISPR / Cas12 gene editing system includes the gene cutting system described above.

[0090] The CRISPR / Cas12 gene editing system can perform gene deletion, insertion, point mutation, base editing, transcriptional activation, transcriptional repression, etc.

[0091] The CRISPR / Cas12 gene editing system may also include conventional reagents used in gene editing in the prior art, but this invention does not impose specific limitations.

[0092] The present invention also provides a recombinant expression vector comprising (i)(ii) or comprising (i)(ii)(iii) of the following: (i) a nucleotide sequence encoding a guide RNA, (ii) a nucleotide sequence encoding a Cas12 hacker nuclease, and (iii) a nucleotide sequence encoding a thioredoxin TrxA.

[0093] In some embodiments of the present invention, the polynucleotide encoding the Cas12 hacker nuclease and the guide RNA are contained in an expression vector.

[0094] In some embodiments of the present invention, the polynucleotide encoding the Cas12 hacker nuclease, the polynucleotide encoding the thioredoxin TrxA, and the guide RNA are contained in one or more expression vectors.

[0095] The present invention also provides a gene cutting method, comprising mixing the gene cutting system described above with a target sequence or cells containing the target sequence to perform gene cutting.

[0096] The present invention also provides a method for genome cutting using the CRISPR / Cas12 gene cutting system, which includes introducing the gene cutting system as described above into cells or viruses containing target sequences for genome cutting.

[0097] The "cells" mentioned in this invention may be bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii; the "viruses" mentioned in this invention may be bacteriophages, such as T4 bacteriophage and T7 bacteriophage.

[0098] This invention also provides a method for in vitro double-stranded DNA cleavage using the CRISPR / Cas12 gene cleavage system, comprising mixing a Cas12 hacker nuclease and a guide RNA (RNP) complex with double-stranded DNA for cleavage. The double-stranded DNA contains a target sequence that can complementaryly pair with a target sequence in the guide RNA, and the target sequence has a PAM sequence upstream of its 5' end. Preferably, the target sequence is a 20 bp DNA fragment following the PAM sequence.

[0099] The preferred reaction temperature for the cutting method is 37°C, and the preferred reaction time is 30 min.

[0100] The present invention also provides a cell obtained by cutting the gene cutting system described above.

[0101] The CRISPR system primarily used in this invention is the CRISPR V system, in which the effector protein is mainly the Cas12 hacker nuclease. Guided by its corresponding guide RNA, the Cas12 hacker nuclease can accurately locate the target gene and cleave the genomic DNA, achieving double-strand breaks.

[0102] The terms “Cas12 hacker”, “Cas12 hacker nuclease”, “Cas12 hacker polypeptide”, “Cas12hacker protein”, and “Cas12 hacker protein” are used interchangeably.

[0103] The terms “single-stranded guide RNA”, “guide RNA”, “sgRNA”, “gRNA” and “chimeric gRNA” are used interchangeably.

[0104] The terms "homology," "identity," or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different polypeptide or nucleic acid molecules. When the same position in the sequence of the compared molecules is occupied by the same base or amino acid in different sequences, then the molecules are homologous at that position. The degree of homology between sequences is determined as a function of the number of shared matching or homologous positions. An "unrelated" or "non-homologous" sequence should have less than 20% homology with one of the sequences disclosed in this invention. A percentage of sequence homology (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) between a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) and another polynucleotide or polynucleotide region (or polypeptide or polypeptide region) means that at the time of alignment, that percentage of bases (or amino acids) are identical in the two sequences being compared. The alignment and percentage homology or sequence identity can be determined using software programs and methods known in the art.

[0105] The term "genomic DNA" refers to the DNA of an organism's genome, including the DNA of bacteria, archaea, fungi, protozoa, viruses, plants, or animals.

[0106] The term "vector" or "expression vector" refers to a replicon, such as a plasmid, bacteriophage, virus, or granule, to which another segment of DNA, or "insertion fragment," can be attached in order to enable the attached segment to replicate within the cell.

[0107] The term "expression cassette" contains a DNA coding sequence operatively linked to a promoter. "Operably linked" means operatively linked, with the components in a relationship that allows them to function in their intended manner. The terms "recombinant expression vector" or "DNA construct" are used interchangeably in this invention to refer to a DNA molecule comprising a vector and at least one insert fragment. Recombinant expression vectors are typically produced for the purpose of expressing and / or amplifying the insert fragment or for constructing other recombinant nucleotide sequences.

[0108] The term "target DNA" refers to a DNA polynucleotide containing a "target site" or "target sequence." The terms "target site," "target sequence," "target protospacer DNA," or "protospacer-like sequence" are used interchangeably in this invention to refer to a nucleic acid sequence present in the target DNA to which the target sequence of the gRNA will bind if sufficient conditions for binding are present. The RNA molecule contains a sequence that binds, hybridizes with, or is complementary to the target sequence within the target DNA, thereby targeting the bound polypeptide to a specific location (target sequence) within the target DNA. "Cleavage" refers to the breakage of the covalent backbone of the DNA molecule.

[0109] The terms "nuclease" and "endonuclease" are used interchangeably to refer to enzymes that have catalytic activity for the degradation of endonucleases for the cleavage of polynucleotides. The "cleavage domain," "active domain," or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within the nuclease that has catalytic activity for DNA cleavage. The cleavage domain may be contained within a single polypeptide chain, or the cleavage activity may arise from the association of two or more polypeptides.

[0110] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0111] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0112] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0113] To better illustrate the purpose, technical solution, and positive effects of this invention, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by the appended claims. For example, developing a Cas12hacker-based single-base editing system by fusing an inactivated Cas12 hacker (e.g., the Cas12 hacker nuclease mutant of this invention) and a base deaminase; developing a Cas12 hacker-based Prime editing system by fusing an inactivated Cas12 hacker and a reverse transcriptase; developing a Cas12 hacker-based transcriptional activation system by fusing an inactivated Cas12 hacker and a transcription activator; developing a Cas12 hacker-based epigenetic modification system by fusing an inactivated Cas12 hacker and a nucleic acid epigenetic modification enzyme; and developing a Cas12 hacker-based transcriptional repression system using an inactivated Cas12 hacker, etc.

[0114] The partial sequences described in this invention are shown below:

[0115] The complete DNA sequence of the wild-type guide RNA containing the targeting sequence is shown below:

[0116] sgRNAv0:

[0117] 5'-GGTTTTGAACTGGGAGAGGTCAAATATCATAATGTTAAGCTAGTTTATCAAAAGCCTA AAAGATTTAAGAAAAAACGCTTGACAAAGTAATAAGATTAGGCTAATATTAAATTACCGCAGTTTGGGCGGGCAGCTAATAATATTAGCTTATGACTTCTAAGGCTTTTGCCTAAAAAGTAAGGGGGAAGGCGACAACCCCCAAAGACCAGCCGGAACTATGGCTGGCAACTATACC CTCTCTTTTGGCATATCAAAGCTAGGGCAAAAACCCCAGACATTCGCCAAAAGCCCAGAACCATGACATTGCAAGAGTTTCGCCCAGTTTCTTTTAAAGATTCAAGCTGATTTAAGCGGCTGAAATGAGATTTTTTAATGCTTGCAACTAAGACTGGGGACTGAGGATAGTTGAAAC TGTCCTCTTCCTCTTTAGCG -3'(SEQ ID NO.46);

[0118] The underlined portion is the target sequence: 5'-TGTCCTCTTCCTCTTTAGCG-3' (SEQ ID NO.49); it is the 20bp fragment following the PAM sequence.

[0119] The Cas12 hacker nuclease Cas12 hacker-1 described in this invention has the following amino acid sequence: (SEQ ID NO.1);

[0120] The nucleotide sequence encoding the Cas12 hacker-1 nuclease includes the following sequence:

[0121]

[0122] The DNA sequences of the backbones of different versions of the guide RNA of the Cas12 hacker-1 nuclease described in this invention are shown below, where light gray represents the tracrRNA sequence; dark gray represents the tracr pair sequence; when there is no linker strand, the tracrRNA sequence + tracr pair sequence constitutes the backbone; when there is a linker strand, the tracrRNA sequence + linker strand + tracr pair sequence constitutes the backbone.

[0123] sgRNAv0:5'-GGTTTTGAACTGGGAGAGGTCAAATATCATAATGTTAAGCTAGTTTATC AAAAGCCTAAAAGATTTAAGAAAAAACGCTTGACAAAGTAATAAGATTAGGCTAATATTAAATTACCGCAGTTTGGGCGGGCAGCTAATAATATTAGCTTATGACTTCTAAGGCTTTTGCCTAAAAAGTAAGGGGGAAGGCGACAACCCCCAAAGACCAGCCGGAACTATGGCTGGCAACTATAC CCTCTCTTTTGGCATATCAAAGCTAGGGCAAAAACCCCAGACATTCGCCAAAAGCCCAGAACCATGACATTGCAAGAGTTTCGCCCAGTTTCTTTTAAAGATTCAAGCTGATTTAAGCGGCTGAAATGAGATTTTTTAATGCTTGCAACTAAGACTGGGGACTGAGGATAGTTGAAAC-3'(SEQ ID NO.50);

[0124] sgRNAv1:5'-AGATTTAAGAAAAAACGCTTGACAAAGTAATAAGATTAGGCTAATATTA AATTACCGCAGTTTGGGCGGGCAGCTAATAATATTAGCTTATGACTTCTAAGGCTTTTTGCC TAAAAAGTAAGGGGGAAGGCGACAACCCCCAAAGACCAGCCGGAACTATGGCTGGCA ACTATACCCTCTCTTTTGgaaaCTTGCAACTAAGACTGGGGACTGAGGATAGTTGAAAC-3' (SEQ ID NO. 51);

[0125] sgRNAv2:5'-ATTACCGCAGTTTGGGCGGGCAGCTAATAATATTAGCTTATGACTTCTAA GGCTTTTGCCTAAAAAGTAAGGGGGAAGGCGACAACCCCCAAAGACCAGCCGGAACTA TGGCTGGCAACTATACCCTCgaaaGAGGATAGTTGAAAC-3' (SEQ ID NO. 52);

[0126] The complete sgRNA sequence is the target sequence TGTCCTCTTCCTCTTTAGCG (SEQ ID NO.49) linked to the 3' end of the aforementioned backbone sequence (SEQ ID NO.50~SEQ ID NO.52). The target sequence is the 20bp fragment after the PAM sequence and can be replaced by other suitable target sequences.

[0127] Example 1: Identification of the Pre-interstitial sequence neighbor motif (PAM) of the Cas12 hacker nuclease

[0128] 1) Construction of p15a-Cas12 hacker_sgRNA_T and p15a-Cas12 hacker_sgRNA_NT

[0129] The nucleotide sequence of the Cas12 hacker-1 nuclease described in this embodiment is shown in SEQ ID NO.44; the nucleotide sequence of the Cas12hacker-2 nuclease is shown in SEQ ID NO.45.

[0130] The specific construction method is as follows:

[0131] p15a plasmid backbone:

[0132]

[0133] Cas12 hacker-1 encoding gene expression cassette and non-coding regions containing target sequences:

[0134] TGTCC TCTTCCTCTTTAGCG GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACccaattattgAACACCCTAACGGGTGTTTTTTTGTTTctggtctacc (SEQ ID NO. 54);

[0135] All components were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The underlined portion represents the target sequence TGTCCTCTTCCTCTTTAGCG (SEQ ID NO. 49), which was assembled into two fragments using Gibson assembly technology. The ligation product was transformed into DH5α competent cells, and single clones were selected for sequencing to identify the inserted sequence, yielding the p15a-Cas12hacker_sgRNA_T plasmid. The p15a plasmid backbone, the Cas12 hacker-encoding gene expression cassette, and the non-coding region not containing the target sequence were then assembled into two fragments to obtain the p15a-Cas12 hacker_sgRNA_NT plasmid. The construction methods for the p15a-Cas12 hacker_sgRNA_T and p15a-Cas12 hacker_sgRNA_NT plasmids corresponding to the Cas12 hacker-2 nuclease were the same as described above.

[0136] 2) Construction of PAM library plasmid pUC19-6N

[0137] The specific construction method is as follows:

[0138] Use primer 6N-F with a 6N random sequence:

[0139] The pUC19 plasmid backbone was extended using circular polymerase cloning with 5'-CCGGCGACGTTGGGTCAACTNNNNNNTGTCCTCTTCCTCTTTAGCGTTTAGAATT TGTC-3' (SEQ ID NO.55) and the common primer 6N-R: 5'-AGTTGACCCAACGTCGC CGG-3' (SEQ ID NO.56). The PCR product was digested with Dpn1 enzyme, and the treated product was transformed into DH5α competent cells. The next day, at least 100,000 single clones were collected from the plate, and the plasmid was extracted. The plasmid library was sequenced to confirm its homogeneity, thus obtaining the PAM library plasmid.

[0140] 3) The procedure for identifying Cas12 hacker nuclease PAM is as follows:

[0141] The corresponding p15a-Cas12 hacker_sgRNA_T and p15a-Cas12hacker_sgRNA_NT plasmids for Cas12 hacker-1 and Cas12 hacker-2, respectively, were transformed into *E. coli* DH5α. The next day, single clones were picked and cultured in seed culture at 37°C. 1 ml of the turbid seed culture was diluted to 100 ml of fresh LB broth. When OD... 600 At a concentration of 0.5, cells were collected by centrifugation. The cells were washed once with sterile water at 4°C and twice with sterile 10% glycerol at 4°C. Finally, the cells were resuspended in 1 mL of sterile 10% glycerol to obtain competent cells. 100 ng of pUC19-6N plasmid was electroporated into DH5α competent cells carrying p15a-Cas12hacker_sgRNA_T and p15a-Cas12 hacker_sgRNA_NT plasmids, respectively. The next day, at least 100,000 single colonies were collected, mixed, and plasmids were extracted to obtain PAM deletion libraries and control libraries. Amplicon sequencing libraries were generated using a two-step amplification method, and the PCR products were purified using VAHTSDNA Clean Beads (Novozymes Biotechnology Co., Ltd.) for Illumina HiSeq sequencing (PE150).

[0142] The raw data was processed to extract 6N random sequence information. The sequence frequencies in the PAM missing library were compared with those in the control library to create a weblogo, thereby obtaining the Preferred Anchor Motif (PAM) information of the Cas12 hacker's preferred anterior interspace sequence.

[0143] Figure 1 The diagram shows the identification process and results of the neighboring motif (PAM) in the pre-septal region of the Cas12 hacker sequence. The results show that both Cas12hacker-1 and Cas12 hacker-2 can effectively identify 5'GTN type PAM sequences (where N represents T, A, G, or C).

[0144] Example 2: Cas12 hacker enables efficient in vitro double-stranded DNA cleavage.

[0145] 1) Preparation of Cas12 hacker-sgRNA RNP complex

[0146] Sumo-Cas12 hacker (SEQ ID NO. 57) and the corresponding target-carrying sgRNAv1 (sgRNA backbone sequence as shown in SEQ ID NO. 51, with the 3' linker to the target sequence as shown in SEQ ID NO. 49) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized fragment was cloned into the *E. coli* expression vector pETDuet. The successfully cloned pETDuet-sumo-Cas12 hacker-sgRNAv1-T plasmid was transformed into *E. coli* BL21(DE3). The next day, single clones were picked and cultured in seed culture at 37°C. The seed culture was then diluted 1:100 to 1L of LB medium. When OD... 600 When the pH reached 0.6, IPTG was added to a final concentration of 0.25 mM, and the protein was induced overnight at 16°C. The bacteria were collected the following day, and after sonication to disrupt the bacterial cells, the protein was purified using a Ni-NTA (GE Healthcare) chromatography column, followed by further purification using a HiLoad 16 / 600 Superdex 200 pg molecular sieve (GE Healthcare). The final protein was stored in a buffer solution of 0.3 M NaCl, 10 mM Tris-HCl, pH 7.5, and 0.5 mM TCEP.

[0147] SEQ ID NO.57:

[0148]

[0149] 2) Preparation of cutting substrate

[0150] The cleavage substrate was a linearized pUC19 plasmid. Using the commercial pUC19 plasmid as a template, the plasmid was amplified by PCR using primers UC-F: 5'-ctgagaatagtgtatgcggcgac-3' (SEQ ID NO.58) and UC-R: 5'-aatgacttggttgagtactcaccag-3' (SEQ ID NO.59). The amplification product was purified by gel extraction to obtain the linearized cleavage substrate.

[0151] 3) The in vitro cleavage assay conditions were as follows: 100 mM NaCl, 10 mM MgCl2, 10 mM Tris-HCl, pH = 7.5. The reaction volume was 20 μL, including 5 nM cleavage substrate and 300 nM Cas12 hacker-sgRNA RNP. The reaction was carried out at 37°C for 30 minutes. After the reaction, 2 μL of 6×Gel Loading Dye (NEB) was added to terminate the reaction. The cleavage products were visualized by 1% agarose gel electrophoresis, as shown below. Figure 2 As shown, the linearized substrate DNA was precisely cleaved into two DNA fragments by the Cas12 hacker nuclease; and its cleavage activity depended on the recognition of the PAM sequence, and its pattern was basically consistent with the adjacent motif (PAM) of the pre-interstitial region sequence obtained above.

[0152] Example 3: Cutting patterns of Cas12 hacker cutting double-stranded DNA

[0153] 1) The Cas12 hacker-sgRNA RNP complex was prepared as described in Example 2.

[0154] 2) Preparation of cleavage substrates. The cleavage substrates consisted of two 56 bp paired-end fluorescently labeled synthetic DNA sequences (synthesized by Sangon Biotech (Shanghai) Co., Ltd.), as follows:

[0155] 56-NTS:5'-TGGGTCAACTgtaTGTCCTCTTCCTCTTAAGCGTTTAcAATTgGaCgaCttcactg-3'(SEQ ID NO.60)

[0156] 56-TS:5'-cagtgaaGtcGtCcAATTgTAAACGCTAAAGAGGAAGAGGACAtacAGTTGACC CA-3'(SEQ ID NO.61)

[0157] The sequence is labeled with a FAM fluorescent group at the 5' end and a Texas Red fluorescent group at the 3' end. Annealing the sequence with dual fluorescent labels and the complementary sequence without dual fluorescent labels at both ends yields a 56 bp cleavage substrate.

[0158] 3) In vitro cleavage experiments were conducted under the conditions of 100 mM NaCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5. The total reaction volume was 80 μL, including 20 nM cleavage substrate and 400 nM Cas12 hacker-sgRNARNP. The total reaction time was 64 minutes, and the reaction temperature was 37 °C. Samples of 10 μL were taken at 0, 1, 2, 4, 8, 16, 32, and 64 minutes, and the reaction was terminated by adding 2×formamide loading buffer. The cleavage products were separated and imaged by 20% TBE-Urea-PAGE.

[0159] Figure 3 This diagram illustrates the cleavage of a 56 bp fluorescently labeled DNA substrate using the Cas12 hacker nuclease and the resulting image. The results show that the Cas12 hacker cleaves the target strand 2 nt below the spacer complementary sequence, while on the non-target strand, it cleaves between positions 13 and 14 of the protospacer. Furthermore, after specific cleavage of the non-target strand, continuous degradation of the non-target strand was observed, eventually forming sticky protruding ends.

[0160] Example 4: Biochemical reaction condition preference of the Cas12 hacker

[0161] 1) The preparation of the Cas12 hacker-sgRNARNP complex and the in vitro cleavage substrate is the same as described in Example 2.

[0162] 2) To determine the optimal reaction conditions for Cas12 hacker in vitro cleavage, the preference of Cas12 hacker for the type and concentration of divalent cations, NaCl concentration, and temperature was investigated. The total reaction time was 60 min. Samples were taken at 0, 5, 10, 20, 40, and 60 min. The reaction products were separated and imaged using 1% agarose gel electrophoresis, and the cleavage efficiency at each time point was obtained by grayscale analysis of the electrophoresis images.

[0163] Figure 4 Figure a shows the temperature preference of the Cas12 hacker nuclease. The optimal temperature for Cas12 hacker to cut DNA is between 30 and 40 degrees Celsius. Figure 4 b-4d shows the Cas12 hacker nuclease's preference for divalent cations. The activity of Cas12hacker depends on Mg.2+ or Mn 2+ The concentration range is 5-20 mM; Figure 4 Figures e and 4f show the NaCl concentration preference of the Cas12 hacker nuclease. The results indicate that in Mg... 2+ Under certain conditions, the optimal salt concentration for Cas12 hacker is 25-100 mM NaCl; under Mn 2+ Under these conditions, the optimal salt concentration for Cas12 hacker is 25-150 mM NaCl.

[0164] Example 5: Electron microscopy sample preparation and collection of the Cas12 hacker-sgRNA-DNA complex.

[0165] 1) Preparation of cryo-electron microscopy samples. The Cas12 hacker-sgRNARNP complex was prepared as described in Example 2. The prepared Cas12 hacker-sgRNARNP was then mixed with substrate DNA at a molar ratio of 1:1.3 in buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP). The ternary complex was then incubated at 25°C for 20 minutes, followed by incubation at 37°C for 10 minutes. Subsequently, the Cas12 hacker-sgRNA-DNA ternary complex was further purified using a Superose 6-increase 10 / 300GL column (GE Healthcare / Cytiva) to concentrate the resulting Cas12 hacker-sgRNA-DNA ternary complex to approximately 3.6 mg / mL. The sequence of the substrate DNA is as follows:

[0166] 33-NTS:5'-TCAACTGTATGTCCTCTTCCTCTTTAGCGCTCA-3'(SEQ ID NO.62)

[0167] 33-TS:5'-TGAGCGCTAAAGAGGAAGAGGACATACAGTTGA-3'(SEQ ID NO.63)

[0168] A glow-discharged mesh (Au 300 mesh, R1.2 / 1.3, ANTcryo, Guangzhou) was discharged with H2 / O2 for 30 s in a Solarus (950) plasma cleaning system (Gatan, USA). Subsequently, 4 μL of the ternary composite was coated onto the glow-discharged mesh. The mesh was then clamped for 3 seconds using a Vitrobot Mark IV (Thermo Fisher Scientific)-1 force clamp at 4 °C and 100% humidity, and the mesh was then rapidly frozen in liquid ethane.

[0169] 2) Collection of cryo-EM data. Cryo-EM data were collected using a 300KV Tatan Krios electron microscope (Thermo Fisher Scientific, USA) equipped with a K3 detector (Gatan, USA). The defocus range was -1.2 to -1.8 μm, and the total dose was 60 electrons / μm. 8004 micrographs of ternary complexes were collected using SerialEM66 at a magnification of 105,000x.

[0170] 3) Processing of cryo-electron microscopy sample collection data. The MotionCor2 algorithm in RELION was used for beam-sensing motion correction of the images, and cryoSPARC was used for subsequent data processing. Contrast transfer function (CTF) parameters were estimated using a patch-based CTF estimation method. Particles were automatically extracted using a blob picker and classified in two dimensions. Particles were then reconstructed from scratch and refined using heterogeneous methods. Selected particles underwent local resolution estimation in cryoSPARC after heterogeneous refinement. DeepEMhancer was used for post-processing to generate sharpened images. Protein and nucleic acid models were manually constructed using COOT and optimized using PHENIX. USCF ChimeraX was used to visualize the cryo-electron microscopy images and atomic models.

[0171] Figure 5 a and 5b are gel images and molecular sieve peak diagrams of the Cas12 hacker-sgRNA-DNA complex preparation process; Figure 5 c is the structural diagram of the Cas12 hacker-TrxA-sgRNA-DNA complex. The resolved structure shows the host factor thioredoxin TrxA, which is tightly bound to the Cas12 hacker nuclease.

[0172] Example 6: The CRISPR-Cas12 hacker system works in conjunction with thioredoxin TrxA to achieve efficient cleavage of the bacterial genome.

[0173] 1) Construction of bacterial in vivo expression plasmids p15a-Cas12 hacker-ec and p15a-Cas12hacker-ab for Cas12 hacker.

[0174] p15a-ec plasmid backbone:

[0175]

[0176] p15a-ab plasmid backbone:

[0177]

[0178] The plasmid backbone sequences and the Cas12 hacker coding gene sequence expression cassette (SEQ ID NO. 44) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Cas12 hacker coding gene sequence expression cassette was assembled with the p15a-ec and p15a-ab plasmid backbones using a two-fragment Gibson assembly. The cells were transformed into *E. coli* DH5α competent cells, plated on LBA plates containing apopramycin, and single colonies were picked for expansion culture. Plasmids were extracted, and after sequencing identification, the p15a-Cas12hacker-ec and p15a-Cas12 hacker-ab plasmids were obtained.

[0179] 2) Construction of the corresponding sgRNA expression plasmid psgRNAv2 for Cas12 hacker.

[0180] pSGKP plasmid backbone:

[0181]

[0182] pSGAB plasmid backbone:

[0183]

[0184] The plasmid backbone and the sgRNAv2 expression cassette containing two Bsa1 sites (SEQ ID NO. 68) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sgRNA expression cassette was assembled with the pSGKP and pSGAB plasmid backbones using a two-fragment Gibson assembly. The cells were transformed into *E. coli* DH5α competent cells, plated on LBA plates containing kanamycin, and single colonies were picked for expansion culture. Plasmids were extracted, and after sequencing identification, the psgRNAv2-ec and psgRNAv2-ab plasmids were obtained. psgRNAv2-ec plasmid backbone and thioredoxin TrxA expression cassette from *E. coli*: agtgtggtagaatatcagcttactattgctttacgaaagcgtatccggtgaaataaagtcaacctttagttggttaatgttacaccaacaacgaaaccaacacgccaggcttattcctgtggagttatatatgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttc The compound tgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctgccactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggcgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggcgtaa (SEQ ID NO. 69) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. It was assembled using a two-fragment Gibson assembly, transformed into competent cells, and the plasmid was extracted and sequenced to obtain psgRNAv2-ec-TrxA.

[0185] 3) Genomic cleavage of *E. coli*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii* was achieved using the Cas12 hacker. Target sequence DNA was inserted into the psgRNAv2-ec, psgRNAv2-ab, and psgRNAv2-ec-TrxA plasmids. In this example, the sequences targeting the *E. coli* and *Klebsiella pneumoniae* genomes are as follows:

[0186] EC-F: 5'-AAACCACCGTCGCGTACTTTACGC-3' (SEQ ID NO.70),

[0187] EC-R: 5'-GGCCGCGTAAAGTACGCGACGGTG-3' (SEQ ID NO.71)

[0188] The sequence targeting the Acinetobacter baumannii genome is as follows:

[0189] AB-F: 5'-AAACtcactatcaattgctggatt-3' (SEQ ID NO.72)

[0190] AB-R: 5'-GGCCaatccagcaattgatagtga-3' (SEQ ID NO. 73).

[0191] These sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. EC-F and EC-R, and AB-F and AB-R were mixed in 1× T4 DNA ligase buffer, and 1 mM ATP and 0.5 μL T4 PNK were added to a final concentration. The mixture was incubated at 37°C for 1 hour. Then, 50 mM NaCl was added to a final concentration, followed by slow annealing. Next, the two target sequence DNAs were inserted into the psgRNAv2-ec and psgRNAv2-ab plasmids, respectively, using Golden Gate assembly. These were then transformed into *E. coli* DH5α competent cells, plated on kanamycin-containing LBA plates, and single colonies were picked for expansion culture. Plasmids were extracted, and after sequencing identification, the psgRNAv2-ec-T, psgRNAv2-ab-T, and psgRNAv2-ec-TrxA-T plasmids were obtained.

[0192] Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii were activated by streaking on LBA plates and incubated overnight upside down at 37°C. Single colonies were picked and inoculated into 100 mL of fresh LB medium and cultured on a shaker at 37°C until the bacterial growth rate reached OD500. 600Once the pH reached 0.5, the bacterial cells were collected by centrifugation. The cells were washed once with pre-cooled sterile water (4°C), then twice with pre-cooled sterile 10% glycerol (4°C). Finally, the bacteria were resuspended in 1 mL of sterile 10% glycerol solution to obtain electrotransformation competent cells. Fresh electrotransformation competent cells were used to electrotransform 100 ng of p15a-Cas12 hacker-ec into *Escherichia coli* and *Klebsiella pneumoniae*, respectively. The p15a-Cas12 hacker-ab plasmid was electrotransformed into competent *Acinetobacter baumannii* cells. The cells were plated on LBA plates containing apopramine and incubated overnight at 37°C. The next day, transformants were picked and inoculated into 100 mL of fresh LB medium. The *E. coli* culture was incubated at 37°C on a shaker until OD reached [value missing]. 600 The bacterial culture was collected by centrifugation after reaching 0.5 μL. The cells were washed once with pre-cooled sterile water at 4°C, then twice with pre-cooled sterile 10% glycerol at 4°C. Finally, the bacteria were resuspended in 1 mL of sterile 10% glycerol solution to obtain electrocompetent cells. The bacterial culture from *Klebsiella pneumoniae* was analyzed at OD... 600 When the concentration reached 0.2, IPTG was added to a final concentration of 1 mM to induce nuclease expression, OD 600 When the concentration reached 0.5, electroporation competent cells were prepared using the same steps as described above. 100 ng of psgRNAv2-ec and psgRNAv2-ec-T plasmids were electroporated into competent cells of *E. coli* and *Klebsiella pneumoniae* containing p15a-Cas12 hacker-ec, respectively. psgRNAv2-ec-TrxA, psgRNAv2-ec-T, and psgRNAv2-ec-TrxA-T plasmids were electroporated into TrxA-deficient *E. coli* containing p15a-Cas12 hacker-ec, and psgRNAv2-ab and psgRNAv2-ab-T plasmids were electroporated into competent cells of *Acinetobacter baumannii* containing p15a-Cas12 hacker-ab. Immediately afterwards, 1 mL of fresh LB medium was added, and the cells were incubated at 37°C for 1.5 hours. The bacterial culture was centrifuged and resuspended in 100 μL of LB medium. The culture was then diluted 10-fold eight times. The diluted culture was spotted onto LBA plates containing kanamycin and apopramycin and incubated upside down overnight at 37°C. The growth of spots on the plates was observed the following day.

[0193] The Cas12 hacker nuclease enables efficient genome cleavage in a variety of bacteria. This example demonstrates the method and process of genome cleavage using live cells of *Escherichia coli*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii*. The process is as follows: Figure 6 As shown in a. Figure 6 b shows the results of genome cleavage in E. coli using the Cas12 hacker nuclease.

[0194] Figure 6c shows the results of genome cleavage in Klebsiella pneumoniae using the Cas12 hacker nuclease. Figure 6 Figure d shows the results of genome cleavage in *Acinetobacter baumannii* using the Cas12 hacker nuclease. The results show that the CRISPR-Cas12 hacker system can effectively cleave the genome in wild-type *Escherichia coli*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii*. However, in *E. coli*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii* lacking thioredoxin TrxA, the cleavage ability of the CRISPR-Cas12 hacker system is reduced. In *E. coli* lacking thioredoxin TrxA, the cleavage ability of the CRISPR-Cas12 hacker system is restored after thioredoxin TrxA is added back.

[0195] Example 7: The CRISPR-Cas12 hacker system, in collaboration with thioredoxin TrxA, enables E. coli to resist bacteriophage infection.

[0196] 1) Construction of the phage infection experiment plasmid p15a-Cas12 hacker-psgRNAv2

[0197] p15a-Cas12 hacker plasmid backbone:

[0198]

[0199] The plasmid backbone and the sgRNAv2 expression cassette containing two Bsa1 sites (SEQ ID NO. 68) were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and assembled using a two-fragment Gibson assembly. The cells were transformed into *E. coli* DH5α competent cells, plated on LBA plates containing apopramine, and single colonies were picked for expansion culture. The plasmid was extracted, and after sequencing identification, the p15a-Cas12 hacker-psgRNAv2 plasmid was obtained. The p15a-Cas12 hacker-psgRNAv2 plasmid backbone and the thioredoxin TrxA expression cassette from *E. coli* (SEQ ID NO. 69) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The cells were assembled using a two-fragment Gibson assembly, transformed into competent cells, and the plasmid was extracted and sequenced to obtain the p15a-Cas12 hacker-psgRNAv2-TrxA plasmid.

[0200] 2) Using Cas12 hacker to resist bacteriophage infection

[0201] Targeted DNA sequences were inserted into the p15a-Cas12 hacker-psgRNAv2 and p15a-Cas12 hacker-psgRNAv2-TrxA plasmids. In this embodiment, the sequences targeting the T4 phage genome are: T4-F1: 5'-AAACaagctactgctctaccagct-3' (SEQ ID NO.75), T4-R1: 5'-GGCCagctggtagagcagtagctt-3' (SEQ ID NO.76); T4-F2: 5'-AAACcggttcagtatcaaaatggg-3' (SEQ ID NO.77), T4-R2: 5'-GGCCcccattttgatactgaaccg-3' (SEQ ID NO.78). These sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. T4-F1 and T4-R1, and T4-F2 and T4-R2 were respectively mixed in 1× T4 DNAligase buffer, and 1 mM ATP and 0.5 μL T4 PNK were added to a final concentration. The mixture was reacted at 37 degrees Celsius for 1 hour. Then, 50 mM NaCl was added to a final concentration, and the mixture was slowly annealed. Next, the two target sequence DNAs were inserted into the p15a-Cas12hacker-psgRNAv2 and p15a-Cas12hacker-psgRNAv2-TrxA plasmids respectively using Golden gate assembly. These plasmids were then transformed into E. coli DH5α competent cells, plated on LBA plates containing apopramine, and single colonies were picked for expansion culture. Plasmids were extracted, and after sequencing and identification, p15a-Cas12 hacker-psgRNAv2-T1, p15a-Cas12 hacker-psgRNAv2-T2, p15a-Cas12hacker-psgRNAv2-TrxA-T1, and p15a-Cas12hacker-psgRNAv2-TrxA-T2 were obtained. The plasmids p15a-Cas12hacker-psgRNAv2, p15a-Cas12 hacker-psgRNAv2-T1, and p15a-Cas12 hacker-psgRNAv2-T2 were transformed into wild-type and TrxA-deficient Escherichia coli BL21(DE3) competent cells, respectively. The plasmids p15a-Cas12hacker-psgRNAv2-TrxA, p15a-Cas12hacker-psgRNAv2-TrxA-T1, and p15a-Cas12 hacker-psgRNAv2-TrxA-T2 were transformed into TrxA-deficient Escherichia coli BL21(DE3) competent cells, respectively.Transformed cells were placed in LB medium supplemented with 50 μg / mL apopramycin and incubated overnight at 37°C with shaking. The next day, 0.3 mL of the overnight culture was mixed with 3 mL of soft LB agar containing 50 μg / mL apopramycin and poured onto a 90 mm² LB agar plate containing 50 μg / mL apopramycin. T4 phage was serially diluted 10-fold in LB medium, and 3 μL of the diluted phage was spotted onto the solidified agar plate. The plates were then incubated at 37°C, and the growth of the phage plaques was observed.

[0202] The CRISPR-Cas12 hacker system can effectively resist phage infection. This embodiment uses T4 phage as an example to demonstrate the method and process of the CRISPR-Cas12 hacker system resisting phage infection, as follows: Figure 7 As shown in a. Figure 7 Figure b shows the results of the CRISPR-Cas12 hacker system's resistance to T4 phage infection. The results indicate that the CRISPR-Cas12 hacker system effectively resists T4 phage infection in wild-type *E. coli*; however, its resistance to T4 phage infection is reduced in *E. coli* lacking thioredoxin TrxA; and in *E. coli* lacking thioredoxin TrxA, the ability of the CRISPR-Cas12 hacker system to resist T4 phage infection is restored upon thioredoxin TrxA supplementation.

[0203] Example 8: Key amino acid sites of the interaction between Cas12 hacker and thioredoxin TrxA

[0204] 1) Structural analysis of the Cas12 hacker-TrxA-sgRNA-DNA complex identified key amino acid residues at the interaction interface between the Cas12 hacker and TrxA. The key amino acid residues for the Cas12 hacker are: W44, Y45, W47, L51, W102, F105, L107, I110, I111, V113, L115, C127, I129, and I172; the key amino acid residues for TrxA are: W32, C33, P35, C36, M38, I61, and I76. Using the p15a-Cas12 hacker-ec plasmid as a template, circular polymerase extension cloning was performed using primers targeting point mutations of these 14 amino acid residues on the Cas12 hacker, resulting in the p15a-Cas12 hacker-ec-mut plasmid with amino acid point mutations. Using psgRNAv2-ec-TrxA plasmid as a template, circular polymerase extension cloning was performed using primers targeting the point mutations of the seven amino acid residues on TrxA to obtain psgRNAv2-ec-TrxA-mut plasmids with amino acid point mutations.

[0205] 2) The method for identifying the effect of mutants on the ability of Cas12 hacker to cut bacterial genomes is the same as described in Example 6.

[0206] Figure 8 Figure a shows a detailed structural diagram of the key amino acid sites involved in the interaction between the Cas12 hacker and thioredoxin TrxA. Figure 8 b represents a comparison of the effects of the Cas12 hacker mutant on bacterial genome cleavage efficiency. Figure 8 c represents a comparison of the effects of TrxA mutants on bacterial genome cleavage efficiency. The results showed that Cas12 hacker mutants W44A, Y45A, W47A, L51A, W102A, F105A, L107A, I110A, I111A, V113A, L115A, C127A, I129A, and C127S, as well as TrxA mutants W32A, C33A, P35A, M38A, I76A, and C33S, all reduced the ability of Cas12 hacker to cleave bacterial genomes to varying degrees.

[0207] Example 9: Key amino acid sites for Cas12 hacker interaction with nucleic acids

[0208] 1) Through structural analysis of the Cas12 hacker-TrxA-sgRNA-DNA complex, the key amino acid residues at the interaction interface between the Cas12 hacker and nucleic acid were identified, including: K137, R138, K149, K150, K156, K159 and K162.

[0209] Using the pETDuet-sumo-Cas12 hacker-sgRNAv1-T plasmid as a template, circular polymerase extension cloning was performed using primers targeting point mutations of these amino acid residues to obtain the pETDuet-sumo-Cas12hacker-sgRNAv1-T-mut plasmid with amino acid point mutations. Subsequently, based on the single-mutant plasmid, three-mutant plasmids (K149A-K150A-K156A), four-mutant plasmids (K137A-R138A-K159A-K162A), and seven-mutant plasmids (K137A-R138A-K149A-K150A-K156A-K159A-K162A) were constructed.

[0210] 2) The preparation of the Cas12 hacker-sgRNARNP mutant protein, the preparation of the cleavage substrate, and the identification of the mutant cleavage activity are the same as those described in Example 2.

[0211] Figure 9 Figure a shows the structural details of the key amino acid sites where the Cas12 hacker interacts with nucleic acids. Figure 9 b is a diagram showing the results of double-stranded DNA cleavage in vitro by the Cas12hacker single mutant. Figure 9 Figure c shows the in vitro cleavage results of Cas12 hacker triple, quadruple, and heptruple mutants on double-stranded DNA. The results show that the Cas12 hacker single mutants K137A, R138A, K149A, K150A, K156A, K159A, and K162A do not affect the cleavage efficiency of double-stranded DNA; the triple mutant has little effect on the cleavage efficiency of double-stranded DNA, while the quadruple and heptruple mutants significantly reduce the Cas12 hacker's ability to cleave double-stranded DNA.

[0212] Example 10: Key amino acid sites for double-stranded DNA cleavage activity of the Cas12 hacker

[0213] 1) Structural analysis of the Cas12 hacker-TrxA-sgRNA-DNA complex identified key amino acid residues in the RuvC domain related to DNA cleavage activity: D378, E478, and D582. Using the pETDuet-sumo-Cas12hacker-sgRNAv1-T plasmid as a template, circular polymerase extension cloning was performed using primers with point mutations targeting these three amino acid residues (the sequences of which are SEQ ID NO: 79-84). Three heterologous expression plasmids of the Cas12 hacker protein with amino acid point mutations were obtained.

[0214] D378-F:(SEQ ID NO:79)

[0215] AAGATTTAAGTGTGGGTATTgctCCCGGAGTACAGGCTGTTGTG;

[0216] D378-R:(SEQ ID NO:80)

[0217] AATACCCACACTTAAAATCTTTGCCTTTAACTATTG;

[0218] E478-F:(SEQ ID NO:81)

[0219] TTATGAACATATTGCCTGGgctGACACGCAGATTAATAATCTACTAAAACAAGTAGAG C;

[0220] E478-R:(SEQ ID NO:82)

[0221] TGTCagcCCAGGCAATATGTTCATAAGTACGAG;

[0222] D582-F:(SEQ ID NO:83)

[0223] TGTAAGTTATTTGACATTCCCCAGCAAGCTgctACTAACGCTGCTAGAAATCATAAGC;

[0224] D582-R:(SEQ ID NO:84)

[0225] AGCTTGCTGGGGAATGTCAAATAAC.

[0226] 2) The preparation of the mutated Cas12 hacker-sgRNARNP and the identification of the mutant cleavage activity were performed in the same way as described in Example 2.

[0227] Figure 10 a is a structural diagram of the Cas12 hacker RuvC structure domain. Figure 10 b shows the comparison between the double-stranded DNA cleavage activity of the three mutants and the wild-type Cas12 hacker protein. The results show that all three mutants can inactivate the Cas12 hacker's ability to cleave double-stranded DNA.

[0228] Example 11 Effect of thioredoxin TrxA on the in vitro double-stranded DNA cleavage activity of Cas12 hacker

[0229] 1) Preparation of Cas12 hacker-sgRNARNP complex without thioredoxin TrxA

[0230] The pETDuet-sumo-Cas12 hacker-sgRNAv1-T plasmid was transformed into *E. coli* BL21(DE3) competent cells lacking TrxA. The next day, single clones were picked and cultured in seed culture at 37°C. The seed culture was then diluted 1:100 to 1 L of LB medium. When OD... 600 When the pH reached 0.6, IPTG was added to a final concentration of 0.25 mM, and the protein was induced overnight at 16°C. The bacteria were collected the following day, and after sonication, the protein was purified using a Ni-NTA (GE Healthcare) chromatography column, followed by further purification using a HiLoad 16 / 600 Superdex 200 pg molecular sieve (GE Healthcare). The final protein was stored in a buffer solution of 0.3 M NaCl, 10 mM Tris-HCl, pH 7.5, and 0.5 mM TCEP.

[0231] 2) Preparation of thioredoxin TrxA

[0232] The thioredoxin TrxA expression cassette (SEQ ID NO. 69) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized fragment was cloned into the E. coli expression vector pET28a. The successfully cloned pET28a-his-TrxA plasmid was transformed into E. coli BL21(DE3). The next day, single clones were picked and cultured in seed culture at 37°C. The seed culture was then diluted 1:100 to 1L of LB medium. Subsequent protein purification steps were as described in Example 2.

[0233] 3) The preparation of the in vitro cutting substrate is the same as described in Example 2.

[0234] 4) To determine the effect of thioredoxin TrxA on the in vitro double-stranded DNA cleavage activity of Cas12 hacker, different concentrations of TrxA protein were added to the purified Cas12 hacker-sgRNARNP complex, which was free of thioredoxin TrxA. The in vitro cleavage reaction conditions were the same as described in Example 2. The concentration of Cas12 hacker-sgRNARNP was 300 nM, and the concentrations of TrxA protein added in a gradient were 0.3 mM, 0.6 mM, 1.2 mM, 2.4 mM, 4.8 mM, and 9.6 mM, respectively.

[0235] Figure 11 The figure shows the effect of thioredoxin TrxA on the in vitro double-stranded DNA cleavage activity of Cas12 hacker. Without TrxA, the in vitro double-stranded DNA cleavage activity of Cas12 hacker is very weak; supplementation with TrxA can enhance the in vitro double-stranded DNA cleavage activity of Cas12 hacker.

[0236] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of the Cas12 hacker nuclease or the polynucleotide encoding the Cas12 hacker nuclease in the preparation of a CRISPR / Cas12 gene cutting system, a CRISPR / Cas12 gene editing system, or in gene cutting or gene editing.

2. The use according to claim 1, characterized in that, The Cas12 hacker nuclease is either wild-type Cas12hacker nuclease or a Cas12 hacker nuclease mutant.

3. The use according to claim 1, characterized in that, The amino acid sequence of the wild-type Cas12 hacker nuclease is shown in any one of SEQ ID NO.1 to 43.

4. The use according to claim 1, characterized in that, The Cas12 hacker nuclease mutant includes any one or more of the following characteristics: 1) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, and the mutation sites are selected from any one or more of the following: W44, Y45, W47, L51, W102, F105, L107, I110, I111, V113, L115, C127, I129 and I172; preferably, the Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1 in any one or more of the following ways: W44A, Y45A, W47A, L51A, W102A, F105A, L107A, I110A, I111A, V113A, L115A, C127A, I129A, I172A, C127S; 2) The Cas12 hacker nuclease mutant is obtained by mutating the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, and the mutation site is selected from any one or more of the following: D378, E478 or D582; preferably, the Cas12 hacker nuclease mutant is obtained by mutating the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1 with any one of the following: D378A, E478A or D582A; 3) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation sites include any one or more of K137, R138, K149, K150, K156, K159, and K162; preferably, the Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, wherein any one of the following mutations is performed: K137A, R138A, K149A, K150A, K156A, or K159A. A single mutation or K149A-K150A-K156A triple mutation, K137A-R138A-K159A-K162A quadruple mutation, K137A-R138A-K149A-K150A-K156A-K159A-K162A heptamutation, or any combination of the above mutations.

5. The use according to claim 1, characterized in that, The gene cutting is extracellular double-stranded DNA cutting or intracellular genome cutting, or the gene editing is selected from gene deletion, gene insertion, point mutation, transcriptional repression, transcriptional activation, or base editing.

6. A CRISPR / Cas12 gene cutting system, characterized in that, The CRISPR / Cas12 gene cutting system includes: 1) Cas12 hacker nuclease or a polynucleotide encoding the Cas12 hacker nuclease; 2) Guide RNA or polynucleotide encoding the guide RNA.

7. The CRISPR / Cas12 gene cutting system according to claim 6, characterized in that, The Cas12 hacker nuclease is the Cas12 hacker nuclease used in any of the applications described in claims 1 to 5.

8. The CRISPR / Cas12 gene cutting system according to claim 6, characterized in that, The guide RNA comprises an RNA backbone sequence and a target sequence. The nucleotide sequence of the backbone sequence is as shown in any one of SEQ ID NO. 50 to 52. The target sequence is a nucleotide sequence complementary to the target sequence in the target gene, preferably a DNA fragment of 20 bp in length following the PAM sequence.

9. The CRISPR / Cas12 gene cutting system according to claim 6, characterized in that, The gene cutting system also includes thioredoxin or a polynucleotide encoding thioredoxin; preferably, the thioredoxin is derived from microorganisms or mammalian cells; more preferably, the thioredoxin is TrxA derived from Escherichia coli; and even more preferably, the amino acid sequence of the thioredoxin TrxA is shown in SEQ ID NO.

47.

10. The CRISPR / Cas12 gene cutting system according to claim 6, characterized in that, The CRISPR / Cas12 gene cutting system is an extracellular gene cutting system.

11. The CRISPR / Cas12 gene cutting system according to claim 10, characterized in that, In the extracellular gene cleavage system, based on the total volume of the gene cleavage system, the concentration ratio of the Cas12 hacker nuclease to the thioredoxin is 1:(0.5-40); preferably, the extracellular gene cleavage system also includes Mg. 2+ or Mn 2+ Preferably, based on the total volume of the gene cutting system, the Mg 2+ or Mn 2+ The concentration is 5-20 mM; preferably, the CRISPR / Cas12 extracellular gene cutting system further includes a pH buffer solution, preferably, the pH of the pH buffer solution is 7.4-7.

6.

12. The CRISPR / Cas12 gene cutting system according to claim 10, characterized in that, The CRISPR / Cas12 extracellular gene cleavage system also includes NaCl; preferably, when the CRISPR / Cas12 extracellular gene cleavage system includes Mg... 2+ At that time, the concentration of NaCl was 25-100 mM; preferably, when the CRISPR / Cas12 extracellular gene cleavage system included Mn 2+ At that time, the concentration of NaCl was 25-150 mM.

13. A CRISPR / Cas12 gene editing system, characterized in that, The CRISPR / Cas12 gene editing system includes the gene cutting system described in any one of claims 6 to 12.

14. A recombinant expression vector, characterized in that, It contains a nucleotide sequence encoding the Cas12 hacker nuclease, as well as a nucleotide sequence encoding the guide RNA and / or a nucleotide sequence encoding thioredoxin.

15. A method for gene cutting or editing, characterized in that, This includes mixing the gene cutting system of any one of claims 6 to 12 or the gene editing system of claim 13 with a target sequence or cells containing the target sequence to perform gene cutting or editing.

16. A cell, characterized in that, The cells are obtained by gene cutting or editing using the gene cutting system of any one of claims 6 to 12 or the gene editing system of claim 13.

17. A Cas12 hacker nuclease mutant, characterized in that, The Cas12 hacker nuclease mutant is selected from any of the following: 1) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, and the mutation sites are selected from any one or more of the following: W44, Y45, W47, L51, W102, F105, L107, I110, I111, V113, L115, C127, I129 and I172; preferably, the Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1 in any one or more of the following ways: W44A, Y45A, W47A, L51A, W102A, F105A, L107A, I110A, I111A, V113A, L115A, C127A, I129A, I172A, C127S; 2) The Cas12 hacker nuclease mutant is obtained by mutating the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, and the mutation site is selected from any one or more of the following: D378, E478 or D582; preferably, the Cas12 hacker nuclease mutant is obtained by mutating the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1 with any one of the following: D378A, E478A or D582A; 3) The Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation sites include any one or more of K137, R138, K149, K150, K156, K159, and K162; preferably, the Cas12 hacker nuclease mutant is obtained by mutation based on the wild-type Cas12 hacker nuclease with the amino acid sequence shown in SEQ ID NO.1, wherein any one of the following mutations is performed: K137A, R138A, K149A, K150A, K156A, or K159A. A single mutation or K149A-K150A-K156A triple mutation, K137A-R138A-K159A-K162A quadruple mutation, K137A-R138A-K149A-K150A-K156A-K159A-K162A heptamutation, or any combination of the above mutations.

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