A novel tranC nuclease and its use

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a newly discovered micro DNA nuclease, which can realize recognition, combination and cutting of specific DNA sequences under the guidance of specific gRNA sequences. The nuclease N3 and N4 which can recognize new PAM sequences and have smaller volume are obtained through the present application, and the editing effect and characteristics of the nuclease in prokaryotic cells and eukaryotic cells are verified, which is expected to expand the application scenarios in the fields of gene editing and gene expression regulation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a novel transposon and CRISPR-Cas12 intermediate (TranC) effector protein obtained through screening and its application in cell gene editing. Background Technology

[0002] Genome editing technology is a core tool in life science research, disease treatment, and biomanufacturing. The CRISPR-Cas system is widely used due to its ease of operation and strong targeting capabilities, but it still faces limitations such as editing efficiency depending on specific PAM (Protospacer Adjacent Motif) sequences, delivery difficulties, insufficient activity in eukaryotic cells, and poor controllability of off-target risks. Currently widely used gene editing tools, Cas9 and Cas12a, require recognition of the PAMs of NNG and TTN, respectively, and both proteins exceed 1000 amino acids in size. Therefore, in practical applications, they still face problems such as PAM limitations (e.g., difficulty in editing A-rich targets) and delivery difficulties. Thus, the gene editing field needs to continuously explore more efficient gene editing tools that can recognize new PAM sequences and are smaller in size.

[0003] Reports indicate that TranC proteins are intermediate proteins possessing both transposon nuclease domains and the sgRNA-binding domain of the CRISPR-Cas12a system, and are evolutionarily closely related to TnpB. For example, the representative LaTranC system can simultaneously use RNA from CRISPR and transposons as guide RNA to recognize corresponding PAMs and cleave double-stranded DNA (WO2023232109A1). TranC proteins have a small amino acid size; therefore, continued exploration and screening of TranC proteins will help to obtain more efficient gene-editing tools that can recognize new PAM sequences and are smaller in size, thereby expanding the application scenarios of gene-editing technology in life science research, disease treatment, and biomanufacturing. Summary of the Invention

[0004] Existing sequence-specific recognition nucleases, particularly CRISPR-Cas system nucleases, have limited recognizable PAM sequence types. In particular, existing technologies lack nucleases targeting A / T nucleotide-rich sites in genomic DNA. This invention provides: 1) A method for screening nucleases with specific PAM sequence preferences; 2) A series of novel TranC nucleases with high editing efficiency obtained through screening using this system; 3) Application methods of the above-mentioned new nucleases in prokaryotic and eukaryotic genome editing. Attached Figure Description

[0005] To better understand the technical solutions described in this invention, the following description is provided in conjunction with the accompanying drawings.

[0006] Figure 1 The editing activity and PAM sequence of each novel nuclease were identified using next-generation sequencing.

[0007] Figure 2 Verify the PAM sequences and editing activities of N3 and N4 proteins in prokaryotes.

[0008] Figure 3 Validate the gene-editing activity of the N3 protein in eukaryotes.

[0009] Figure 4 Validate the gene-editing activity of the N4 protein in eukaryotes. Detailed Implementation

[0010] The present invention provides a nuclease, characterized in that the amino acid sequence of the nuclease is as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 3 or SEQ ID NO: 4, and retains the biological function of the sequence from which it originated.

[0011] Another aspect of the present invention provides a fusion protein, characterized in that the fusion protein contains the aforementioned nuclease domain.

[0012] Preferably, the fusion protein further comprises at least one other functional domain that is directly or operatively linked to the N-terminus or C-terminus of the nuclease.

[0013] Preferably, the functional domain is selected from one or more of the following: deaminase domain, recombinase domain, signal peptide, cell-penetrating peptide, reverse transcriptase domain, epigenetic modifying enzyme domain, and fluorescent protein domain.

[0014] Another aspect of the present invention provides an isolated polynucleotide, characterized in that the polynucleotide is a polynucleotide sequence encoding the aforementioned nuclease or encoding the aforementioned fusion protein.

[0015] Another aspect of the present invention provides a vector, characterized in that the vector comprises the aforementioned polynucleotide and a regulatory element operatively connected thereto.

[0016] Another aspect of the present invention provides a gene editing system, characterized in that the system includes the aforementioned nuclease and at least one gRNA; the gRNA includes a backbone sequence capable of binding to the nuclease of the present invention and a spacer sequence capable of targeting a target sequence.

[0017] The aforementioned gene editing system is characterized in that, i) The amino acid sequence of the nuclease is as shown in SEQ ID NO: 3, or has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3, and retains the biological function of its derived sequence; the gRNA has the nucleotide sequence as shown in SEQ ID NO: 13, wherein consecutive Ns represent spacer sequences that bind to the target sequence, and the remainder is the backbone sequence; or ii) The amino acid sequence of the nuclease is as shown in SEQ ID NO:4, or has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:4, and retains the biological function of the sequence from which it is derived. The gRNA has the nucleotide sequence as shown in SEQ ID NO:16, wherein consecutive Ns represent spacer sequences that bind to the target sequence, and the remainder is the backbone sequence.

[0018] Another aspect of the present invention provides a composition, characterized in that the composition comprises: (i) a protein component selected from: the nuclease of the present invention or the fusion protein of the present invention; (ii) a nucleic acid component, which is gRNA, the gRNA comprising a backbone sequence capable of binding the nuclease of the present invention and a spacer sequence capable of targeting a target sequence; the gRNA having a nucleotide sequence as described in SEQ ID NO: 11-16, wherein consecutive Ns represent the spacer sequence binding to the target sequence, and the remainder is the backbone sequence; the protein component and the nucleic acid component bind to each other to form a complex.

[0019] The aforementioned composition is characterized in that: i) The amino acid sequence of the nuclease is as shown in SEQ ID NO: 3, or has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3, and retains the biological function of its derived sequence; the gRNA has the nucleotide sequence as shown in SEQ ID NO: 13, wherein consecutive Ns represent spacer sequences that bind to the target sequence, and the remainder is the backbone sequence; or ii) The amino acid sequence of the nuclease is as shown in SEQ ID NO:4, or has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:4, and retains the biological function of the sequence from which it is derived. The gRNA has the nucleotide sequence as shown in SEQ ID NO:16, wherein consecutive Ns represent spacer sequences that bind to the target sequence, and the remainder is the backbone sequence.

[0020] Another aspect of the present invention provides an engineered host cell, characterized in that the host cell comprises the aforementioned nuclease, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned gene editing system, or the aforementioned composition.

[0021] The aforementioned nuclease, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned gene editing system, or the aforementioned composition, or the aforementioned host cell, in the application of gene editing, gene targeting, or gene cutting; or, in the preparation of reagents or kits for gene editing, gene targeting, or gene cutting.

[0022] The aforementioned nuclease, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned gene editing system, or the aforementioned composition, or the aforementioned host cell may be used in the following applications: targeting and / or editing target nucleic acids; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; non-specifically cleaving and / or degrading side-branched nucleic acids; non-specifically cleaving single-stranded nucleic acids; nucleic acid detection; specifically editing double-stranded nucleic acids; base editing double-stranded nucleic acids; base editing single-stranded nucleic acids; targeted insertion of nucleic acid fragments; targeted deletion of nucleic acid fragments.

[0023] the term As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0024] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, in the description of a specific polypeptide amino acid sequence in this application, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0025] As used in this article, “genome” encompasses not only chromosomal DNA located in the cell nucleus, but also organelle DNA located in subcellular components of the cell, such as mitochondria and plastids.

[0026] As used herein, “organism” includes any organism suitable for genome editing, preferably eukaryotes or prokaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; microorganisms such as Escherichia coli and Bacillus subtilis; and plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, and Arabidopsis thaliana.

[0027] As used herein, "genetically modified organism" or "genetically modified cell" means an organism or cell whose genome contains exogenous polynucleotides or modified genes or expression regulatory sequences. For example, exogenous polynucleotides can be stably integrated into the genome of an organism or cell and inherited across generations. Exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. Modified genes or expression regulatory sequences are sequences in the genome of an organism or cell that contain single or multiple deoxynucleotide substitutions, deletions, and additions.

[0028] In relation to a sequence, “exogenous” means a sequence that originates from a foreign species, or, if from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0029] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, and “T” for deoxythymidine. Although nucleotide sequences may be represented as DNA sequences (including) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0030] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0031] As used herein, methods for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0032] As used herein, "vector" or "expression vector" refers to a tool suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0033] As used herein, a “vector” can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0034] As used herein, a "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in a cell, regardless of whether it originates from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0035] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0036] Codon optimization refers to the modification of nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways.

[0037] As used herein, "introducing" nucleic acid molecules (e.g., plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into an organism refers to transforming the organism's cells with the nucleic acid or protein, enabling the nucleic acid or protein to function within the cell. The term "transformation" as used in this invention includes both stable and transient transformations.

[0038] Example A further understanding of the invention can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Obviously, various modifications and variations can be made to the invention without departing from its spirit; therefore, such modifications and variations are also within the scope of protection claimed in this application.

[0039] Example 1: PAM reduction experiment of TranC nuclease 1.1 Candidate Proteins This study used bioinformatics methods to mine and screen a series of potentially active novel RNA-guided TranC nucleases from prokaryotic genome and metagenomic data in databases such as NCBI, JGI, and Mgnify (for specific mining methods, refer to WO2023232109A1 and Jin S, Zhu Z, Li Y, et al. Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons[J]. Cell, 2025, 188(22): 6283-6300., which are incorporated herein by reference). Four of these proteins were verified and identified. The amino acid sequence length of these four proteins is less than 600 amino acids, belonging to compact CRISPR effector proteins (as shown in Table 1).

[0040] Table 1 Candidate Proteins

[0041] 1.2 Carrier Construction 1) Construct the pEffect plasmid, which contains candidate proteins N1-N4 and gRNA locus sequences, along with the ampicillin-resistant prokaryotic expression vector pEffector. Expression is driven by the proC+SD4 promoter, and the vectors are named pN1, pN2, pN3, and pN4, respectively. The gRNA targets the target site on the pTarget plasmid.

[0042] 2) Construct the pTarget plasmid containing a plasmid library pTarget-nnnnn (where n is selected from A, T, G, C) that provides spectinomycin resistance to the gRNA binding target. The 5' end of this plasmid library contains a 5-nt random sequence upstream of the target as a potential PAM region to verify whether candidate nucleases can recognize and cleave specific PAM sequences. If the candidate protein can recognize and cleave the target of a specific PAM sequence, the pTarget vector carrying that specific PAM sequence will be selectively eliminated from the bacterial population, resulting in a decrease in the number of that PAM sequence observed in subsequent next-generation sequencing results.

[0043] 1.3 Co-conversion pN1-pN4 plasmids were transformed into *E. coli*, and the transformed *E. coli* cells were then prepared as electroporation competent cells. The pTarget-nnnnn plasmid library was electroporated into each competent cell containing the respective plasmid. After 1 hour of recovery, the recovery fluid was plated on LB agar plates containing ampicillin and spectinomycin (amp+Spe) and incubated overnight at 37°C. The next day, *E. coli* colonies were picked, and plasmids were extracted. At this point, plasmids that had already been cleaved by candidate nucleases in the plasmid library had been eliminated.

[0044] 1.4 Plasmid Analysis The plasmid library extracted in 1.3 was amplified by PCR using target-specific primers: (upstream: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNcgccctctggtaaggttggg; downstream: TGGAGTTCAGACGTGTGCTCTTCCGATCTggcatcagagcagccgattgtc), and the amplified products were then subjected to next-generation sequencing.

[0045] 1.5 PAM Sequence Analysis In the next-generation sequencing results of plasmids, the abundance percentage (or base distribution) of A, T, G, and C bases in the PAM sequence was statistically analyzed, and the abundance percentage in the original pTarget plasmid was used as the control group. Specifically, a 5-nt random sequence n1n2n3n4n5 was constructed at the 5' end upstream of the target site. Nucleases can recognize potential PAM sequences and cleave them at the 3' end of the PAM sequence. Therefore, potential PAM sequences can be predicted by detecting the base sites where the abundance decreases after cleavage. The results are as follows: Figure 1As shown, the experimental results indicate that the A base content at certain sites in the N3 and N4 proteins is decreased, suggesting that these are potential PAM sequences. Specifically, the N3 protein may recognize the 5'-AAAA-3' PAM sequence (the last position of the PAM sequence is a nucleotide linked to the genomic target site), while the N4 protein may recognize the 5'-AAN-3' PAM sequence. No obvious PAM sequence preference was detected in the N1 and N2 proteins.

[0046] Example 2: PAM sequence verification of N3 and N4 proteins To further verify the identified PAM sequence, pTarget plasmids containing spectinomycin resistance and different PAM sequences were electroporated into N3 and N4 competent cells, respectively. The results showed that N3 nuclease efficiently cleaved plasmids containing the AAAA PAM sequence; while N4 nuclease efficiently cleaved plasmids containing AAA, AAT, AAG, or AAC PAM sequences, but could not cleave plasmids containing the TAT PAM sequence, indicating that N4 recognizes the AAN PAM sequence. Figure 2 This embodiment further confirms that the N3 and N4 proteins of the present invention have significant nuclease activity and recognition sequence specificity.

[0047] Example 3: Editing effects of N3 and N4 in eukaryotic cells and screening of gRNA backbone structures To further investigate the nuclease activities of N3 and N4 proteins in eukaryotic cells and optimize their editing efficiency, this embodiment verifies the eukaryotic nuclease activities of the N3 and N4 proteins screened in the previous embodiments. HEK293T cells were used for verification, and the target genes were HEK3 (GenBank: AL359552.16), RNF2 (GenBank: AL109865.36), and RUNX1 (NCBI NG_011402.2). The target sequences are shown in Table 2. Specifically, various gRNAs were designed for N3, N4, and the target genes (see Table 3). Each gRNA included a backbone sequence and a spacer sequence, where consecutive N's indicated the spacer sequence binding to the target sequence, and the remaining portion was the backbone sequence. The N3 / N4 proteins and their corresponding gRNAs were transfected into HEK293T cells. After 72 hours of transfection, DNA was extracted, and next-generation sequencing was used to detect the editing efficiency of the target genes. The results showed that the N3 protein could achieve gene editing at all three endogenous targets. Specifically, when the N3 protein was used in combination with gRNA-V3 (SEQ ID NO: 13), the editing efficiency at the RUNX1 target reached 2.5%, while the editing efficiency was below 1% when used with gRNA-V1 or V2. Figure 3 ).

[0048] Table 2 Validation Target Information

[0049] Table 3 gRNA Design

[0050] For the N4 protein, when used in conjunction with gRNA-V3 (SEQ ID NO: 16), efficient gene editing can be achieved at three endogenous targets, with an editing efficiency of up to 36.7% at the RUNX1 site. However, when used in conjunction with gRNA-V1 and gRNA-V2, almost no editing efficiency is achieved. Figure 4 ).

[0051] The experimental results above demonstrate that the newly discovered nucleases N3 and N4, when paired with corresponding optimized gRNAs, can achieve efficient gene editing in eukaryotes.

[0052] The above descriptions of the embodiments are merely illustrative of the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the present invention. Sequence List: >SEQ ID NO:1 N1 protein MKEKKERFVLKLPLKTQKYQEDIIDKRLEISRRMYNQLVAKTTNEYDQMIKTRRYRGIREELSQIYDSKTEEEKSAKKRSSRERELYQMLNDMYKEYGFTDFSMRALANNQRQPFKKNIDSATATKIGYRLWTSWDRFLYGDGRAVHFCRYGNYNSVEGSSNKSGIRIKKDAISKKNVWSGTMPLALLWNGLEIPVVVKNIYEKEALECEVAFNRIIRETVRGKSKYYLQIVLKGKPPIKYNEKTGELKHPIGEGRVGIDLGLQTLAAVCENEVKLVPLASRVRTKDIEEQKAELQQKMDRSRRAMNPDNYNEDGTIKKQGAKKVIWVKSNHYKQMQLQLKELERKQAAVRKYQHYVLVNELLAMGNQFYVVDKNYKQMQERKDLKQKEDGSYESRKNYGKVINEKGPAMLLNLLSQKLEQCDGEYTKVSAASKICQYSHISDEYQKKETAERYDAASGCQKNLYVAFLLTHMIDATELDKAGCKESFETFKKMHDALFDQMRQQKESGTKFPASMGI >SEQ ID NO:2 N2 protein * >SEQ ID NO:3 N3 protein MATTDKKDEDKEKLRAYKFRLDPNQAQTTALYQAAGAARYTYNMLTAYNLEVNRLRDEYWKKRHDEGISDADIKKELNALAKDDKRYKQLKYGTFSTQYLTPERDRHKDAALRIKNGEDPVVIWNQETERSANPWL HTVNRRVLVSGLQNASDAWDNFWDSRMGKRAGRLVGTPRFKKKGVSRDSFTVPAAKEKEMGAYGAAYLRGEPYKQGRRKITDYRHVRLSYLGTIRTFNSTKPLVKAVAAGAKIRSYTVSRNADRWYVSFLVKFS EPIRRSATKRARAAGSVGVDLGVKYLASLSDSEAPQRFPNLKFVEGLPSLENPRWSGTSSRRLRLKLQRALARSQKGSNRRSRLVKQIARLHHMTALRRESNLHQLTKKLATGYTLVGFEDLNVSGMTASAKGTVENPGKNVAQKSGLNRVVLDAAFGVFRNQLEYKAVWYGSAFEKVNRYFASSQTCSECGRKAKTKLTLRDRVFDCVYCGNMMDRDFNAAVNICREAQRLFDEKLASEDGESLNGRGSRGALRGAKTVEASRPPASHRRGSP >SEQ ID NO:4 N4 protein MAKKTSTEAADTTLRAYKLRLDPTQAQATALAQCAGAARYAYNLLTAHNLEVSRTRAGYWHARIDAGADEATVKAELKALAQSDPATYKALGFQAYATTHLTPEIMRHRAAGKAIESGADPATVWPETERSSEPWMHTIPRRVLVSGLQSADKAWKNFFDSRTGARAGRLVGTPRFKRKGASRDSFTIPAPETIGAYGTAYLRGEPEYARRKAQLKRRGIKTTPTIEDYRHVRLSHLGVIRTHDTTRHLVKAVRAGAQVRSYTVSRAANRWYVSILVELTRASASPTRAQRSAGAVGVDLGVRYLAALSDEQAPQRFAQYPSLEFTGDGAPTLANPRWARTAEKRLVRLQRALSRAQKGSNRRARIVQQIARHHHLVALRRESGLHQVSKRLSTGYTLIGLEDLAVAGMTASAAGTVEAPGKNVRQKAGLNRSILDAAFSTLRRQLEYKASWYGSQVQIIDRFFASSQTCSACGARAKTKLGLHVRVFECAACGARIDRDVNAARNIRAEAVRMYEAQLAPGTGESLNGRGAAGSDAAGSVVLEDAALDASRPAATGGGSP >SEQ ID NO:5 N1 gRNA locus sequence >SEQ ID NO:6 N2 gRNA locus sequence >SEQ ID NO:7 N3 gRNA locus sequence GAGAAGGTTAACCGTTATTTTGCTTCGTCGCAGACGTGTTCGGAGTGCGGGAGGAAAGCGAAAACCAAGCTGACTCTGCGCGACCGTGTCTTCGACTGTGTGTACTGTGGGAATATGATGGATCGTGATTTTAACGCGGCCGTCAATATTTGCCGTGAGGCGCAGAGGCTTTTTGACGAAAAACTCGCCTCAGAAGATGGGGAGAGTCTAAACGGACGTGGAAGTCGAGGCGCTCTTCGGGGTGCTAAGACTGTAGAAGCGTCAAGACCACCTGCGAGTCATCGTAGAGGGTCACCGTAGATGAGTAATCGTCTACCCATCCATTGCATTATGCACGCAACTGAATAGAGTTTTGCAGACATGCGGACGTTATACCTAGTCAGCAAGCGTCTGCCCCGCGTGTGCATGGGTGGTTCCAGCAAGGAACCCCACAGCACCGAATCATCACGGTCTGCCCCGCGTGTGCATGGGTGGTTCCCCTCTCATCAGTGACCATCTCAGGAATTTTGAGTCTGCCCCGCGTGTGCATGGGTGGTTCCA >SEQ ID NO:8 N4 gRNA locus sequence TTCTTTGCATCTTCTCAGACGTGTAGCGCTTGCGGAGCACGAGCGAAAACCAAGCTCGGACTCCACGTGCGTGTCTTTGAGTGCGCAGCCTGCGGTGCGCGGATTGATCGTGACGTGAATGCGGCACGTAATATCCGCGCGGAGGCTGTACGGATGTATGAGGCGCAACTCGCCCCCGGCACGGGGGAGAGTCTAAACGGACGCGGAGCCGCGGGCTCTGATGCTGCCGGTTCGGTGGTGTTGGAGGATGCGGCGTTGGATGCGTCAAGGCCAGCCGCCACGGGCGGCGGGTCACCGTAGACGAGTAATCGTCTACTCATCCCTTTATATGTATACGCGTGGAAACTAAATAATTTTTGATTCTTGGCTGGGTATCGTGCCTTGTCAGCAAGCATGTTCCCCGTATGCGCGGGAATGAGCCGGAATACCTTCCCGAGCTGAAAGAAGCTTCGAAATGTTCCCCGTATGCGCGGGAATGAGCCGGAGCCCAAGAACAGCGCGAGGATAATGCCAATGTGTTCCCCGCGTGTGCGGGGATGAGCCCTTTCCCCCTATAAGTTAACCATGTTCCCCGCGTGTGCGGGGATGAGTCCACGAGGTCACTCTATACAGAGTGACCCCAGGGGTTTTCTCTGCGTGTGCGGGAATGAGCCGTCTGACGATAAGAAGAAGGGGGTCCGCGCGACGTGTTCCTTGCGTGAGTGGGGATGAGCCT >SEQ ID NO:9 upstream primer sequence ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNcgccctctggtaaggttggg >SEQ ID NO:10 downstream primer sequence TGGAGTTCAGACGTGTGCTCTTCCGATCTggcatcagagcagccgattgtc >SEQ ID NO:11 N3 gRNA-V1 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) CTCGCCTCAGAAGATGGGGAGAGTCTAAACGGACGTGGAAGTCGAGGCGCTCTTCGGGGTGCTAAGACTGTAGAAGCGTCAAGACCACCTGCGAGTCATCGTAGAGGGTCACCGTAGATGAGTAATCGTCTACCCATCCATTGCATTATGCACGCAACTGAATAGAGTTTTGCAGACATGCGGACGTTATACCTAGTCAGCAAGCGTCTGCCCCGCGTGTGCATGGGTGGTTCCANNNNNNNNNNNNNNNNNNNN >SEQ ID NO:12 N3 gRNA-V2 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) CTCGCCTCAGAAGATGGGGAGAGTCTAAACGGACGTGGAAGTCGAGGCGCTCTTCGGGGTGCTAAGACTGTAGAAGCGTCAAGACCACCTGCGAGTCATCGTAGAGGGTCACCGTAGATGAGTAATCGTCTACCCATCCATTGCATTATGCATGGGTGGTTCCANNNNNNNNNNNNNNNNNNNN >SEQ ID NO:13 N3 gRNA-V3 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) GAGGCTTTTTGACGAAAAACTCGCCTCAGAAGATGGGGAGAGTCTAAACGGACGTGGAAGTCGAGGCGCTCTTCGGGGTGCTAAGACTGTAGAAGCGTCAAGACCACCTGCGAGTCATCGTAGAGGGTCACCGTAGATGAGTAATCGTCTACCCATCCATTGCATTATGCACGCAaaggTGCCCCGCGTGTGCATGGGTGGTTCCANNNNNNNNNNNNNNNNNNNN >SEQ ID NO:14 N4 gRNA-V1 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) CTCGCCCCCGGCACGGGGGAGAGTCTAAACGGACGCGGAGCCGCGGGCTCTGATGCTGCCGGTTCGGTGGTGTTGGAGGATGCGGCGTTGGATGCGTCAAGGCCAGCCGCCACGGGCGGCGGGTCACCGTAGACGAGTAATCGTCTACTCATCCCTTTATATGTATACGCGTGGAAACTAAATAATTTTTGATTCTTGGCTGGGTATCGTGCCTTGTCAGCAAGCATGTTCCCCGTATGCGCGGGAATGAGCCGNNNNNNNNNNNNNNNNNNNN >SEQ ID NO:15 N4 gRNA-V2 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) CTCGCCCCCGGCACGGGGGAGAGTCTAAACGGACGCGGAGCCGCGGGCTCTGATGCTGCCGGTTCGGTGGTGTTGGAGGATGCGGCGTTGGATGCGTCAAGGCCAGCCGCCACGGGCGGCGGGTCACCGTAGACGAGTAATCGTCTACGCATGTTCCCCGTATGCGCGGGAATGAGCCGNNNNNNNNNNNNNNNNNNNN >SEQ ID NO:16 N4 gRNA-V3 (where NNNNNNNNNNNNNNNNNNNN represents the spacer sequence) CGGATGTATGAGGCGCAACTCGCCCCCGGCACGGGGGAGAGTCTAAACGGACGCGGAGCCGCGGGCTCTGATGCTGCCGGTTCGGTGGTGTTGGAGGATGCGGCGTTGGATGCGTCAAGGCCAGCCGCCACGGGCGGCGGGTCACCGTAGACGAGTAATCGTCTACTCATCCCTTTATATGTATACGCGTGGAAaaggTTCCCCGTATGCGCGGGAATGAGCCGNNNNNNNNNNNNNNNNNNNN >SEQ ID NO:17 HEK3 Target 1 (N3) AAAA GCATCAAGCCTACAAATGCA >SEQ ID NO:18 RNF2 target 1 (N3) AAAA CATGGGAACTCAGTTTATAT >SEQ ID NO:19 RUNX1 Target 1 (N3) AAAA GAAATCATTGAGTCCCCCGC >SEQ ID NO:20 HEK3 target 1 (N4) AAT GCATGCTTACTTGCAGCAGA >SEQ ID NO:21 RNF2 target 1 (N4) AAT AAAGAAGAGAGAAAAACAAT >SEQ ID NO:22 RUNX1 Target 1 (N4) AAT CATTGAGTCCCCCGCCTTCA >SEQ ID NO:23 pTarget sequence: >SEQ ID NO:24 pEffector-N1 >SEQ ID NO:25 pEffector-N2 >SEQ ID NO:26 pEffector-N3 >SEQ ID NO:27 pEffector-N4

Claims

1. A nuclease, characterized in that, The amino acid sequence of the nuclease is shown in SEQ ID NO:

4.

2. An isolated polynucleotide, characterized in that, The polynucleotide is a polynucleotide sequence encoding the nuclease of claim 1.

3. A carrier, characterized in that, The vector comprises the polynucleotide of claim 2 and a regulatory element operatively linked thereto.

4. A gene editing system, characterized in that, The system comprises the nuclease of claim 1 and at least one gRNA; The gRNA comprises a backbone sequence capable of binding to the nuclease of claim 1 and a spacer sequence capable of targeting the target sequence. The backbone sequence of the gRNA is shown as bases 1-224 of SEQ ID NO:

16.

5. The gene editing system according to claim 4, characterized in that, The nucleotide sequence of the gRNA is shown in SEQ ID NO: 16, where consecutive Ns represent the spacer sequence that binds to the target sequence, and the remaining part is the backbone sequence.

6. A composition, characterized in that, The composition comprises: (i) a protein component selected from the nuclease of claim 1; and (ii) a nucleic acid component, which is gRNA, the gRNA comprising a backbone sequence capable of binding to the nuclease of claim 1 and a spacer sequence capable of targeting a target sequence; wherein the backbone sequence of the gRNA is shown as bases 1-224 of SEQ ID NO:16; the protein component and the nucleic acid component bind to each other to form a complex.

7. The composition according to claim 6, wherein the nucleotide sequence of the gRNA is as shown in SEQ ID NO: 16, wherein consecutive Ns represent spacer sequences that bind to the target sequence, and the remainder is a backbone sequence.

8. An engineered host cell, characterized in that, The host cell comprises the nuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the gene editing system of claim 4 or 5, or the composition of claim 6 or 7.

9. The non-therapeutic use of the nuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the gene editing system of claim 4 or 5, or the composition of claim 6 or 7, or the host cell of claim 8 in gene editing, gene targeting, or gene cutting.

10. Use of the nuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the gene editing system of claim 4 or 5, or the composition of claim 6 or 7, or the host cell of claim 8 in the preparation of reagents or kits for gene editing, gene targeting, or gene cutting.

11. The non-therapeutic use of the nuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the gene editing system of claim 4 or 5, or the composition of claim 6 or 7, or the host cell of claim 8 in targeting and / or editing target nucleic acids.

12. Use of the nuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the gene editing system of claim 4 or 5, or the composition of claim 6 or 7, or the host cell of claim 8 in the preparation of reagents or kits for targeting and / or editing target nucleic acids.

13. The application of claim 11 or the use of claim 12, wherein the targeting and / or editing of the target nucleic acid is the cleavage of double-stranded DNA, single-stranded DNA or single-stranded RNA.

14. The application of claim 11 or the use of claim 12, wherein the targeting and / or editing of the target nucleic acid is nonspecific cleavage and / or degradation of the side branch nucleic acid.

15. The application of claim 11 or the use of claim 12, wherein the targeting and / or editing target nucleic acid is a non-specific cleavage of a single-stranded nucleic acid.

16. The application of claim 11 or the use of claim 12, wherein the targeted and / or edited target nucleic acid is a nucleic acid detection.

17. The application of claim 11 or the use of claim 12, wherein the targeting and / or editing of the target nucleic acid is the specific editing of a double-stranded nucleic acid.

18. The application of claim 11 or the use of claim 12, wherein the targeted and / or edited target nucleic acid is a base-edited double-stranded nucleic acid or a base-edited single-stranded nucleic acid.

19. The application of claim 11 or the use of claim 12, wherein the targeting and / or editing of the target nucleic acid is a targeted insertion of a nucleic acid fragment or a targeted deletion of a nucleic acid fragment.

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