Cas mutant proteins with improved editing activity and uses thereof

Abstract

The application discloses a Cas mutant protein with improved editing activity and application thereof. The Cas mutant protein comprises at least one amino acid mutation compared with the protein shown in SEQ ID No. 1; the amino acid mutation of the Cas mutant protein comprises any one or more of the following: the 5th amino acid of SEQ ID No. 1 is mutated into lysine, the 7th amino acid is mutated into lysine, the 396th amino acid is mutated into lysine, the 397th amino acid is mutated into lysine, the 462th amino acid is mutated into arginine, the 494th amino acid is mutated into lysine, the 599th amino acid is mutated into lysine and the 788th amino acid is mutated into serine. The application takes a known protein as a parent, carries out site-directed mutagenesis on amino acid residues that may play a key role, and obtains various single-point mutants and combination mutants. The Cas mutant protein provided by the application has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a Cas mutant protein with enhanced editing activity and its applications. Background Technology

[0002] The CRISPR-Cas system has rapidly developed as a powerful genome editing tool, making significant contributions to biomedical research, gene therapy, and agricultural breeding. While the mature type II Cas9 system remains widely used, a large number of previously uncharacterized CRISPR-Cas systems have been identified, revealing their diverse potential in genome engineering. Among them, type V CRISPR-Cas systems (such as Cas12i) are considered to have promising applications due to their small protein size, unique PAM requirements, low mismatch tolerance, and ability to independently process CRISPR-RNA (crRNA) for multiple targeting.

[0003] Cas12i is an RNA-guided endonuclease that achieves site-specific DNA cleavage by forming a crRNA-guided R-loop structure at the target site. Cas12i's recognition of target DNA depends on the protospacer adjacent motif (PAM) adjacent to the target sequence, typically 5'-TTN-3'. This PAM serves as the initial docking signal for R-loop formation, ensuring high-fidelity targeting. However, this strict PAM dependence also limits the genomic range that Cas12i can target to some extent.

[0004] Although recent engineering modifications to Cas9 and Cas12 proteins have somewhat relaxed their PAM recognition requirements, such modifications often come with the risk of decreased cleavage efficiency or increased off-target effects. Therefore, developing Cas effectors that can better balance accuracy, efficiency, and programmability in different biological systems remains of great importance.

[0005] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Cas mutant protein with enhanced editing activity and its application, aiming to solve the problem that the editing activity of existing Cas proteins needs to be improved.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a Cas mutant protein with enhanced editing activity, said Cas mutant protein comprising at least one amino acid mutation compared to the protein shown in SEQ ID No. 1; The Cas mutant protein contains any one or more of the following amino acid mutations: mutating the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, and the 788th amino acid to serine.

[0008] Optionally, compared with the protein shown in SEQ ID No. 1, it contains one amino acid mutation, wherein the amino acid mutation is to mutate the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, or the 788th amino acid to serine.

[0009] Optionally, compared to the protein shown in SEQ ID No. 1, it contains multiple amino acid mutations, and the amino acid mutations are selected from any of the following: (A) The fifth amino acid is mutated to K, and mutations also exist at other amino acid sites; (B) The 397th amino acid is mutated to K, and mutations also exist at other amino acid sites; (C) The 599th amino acid is mutated to K, and mutations also exist at other amino acid sites; Specifically, the mutation at position 5 of (A) is K, and mutations also exist at other amino acid sites, including: (A1) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A2) The 5th position mutates to K, the 7th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A3) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A4) The 5th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A5) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A6) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 369th position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A7) The 5th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A8) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 944th position mutates to R; (A9) The 5th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (A10) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, and the 601st position mutates to R; (A11) The 5th position mutates to K, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A12) The 5th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A13) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A14) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A15) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A16) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A17) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A18) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A19) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A20) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; The mutation at amino acid position 397 in (B) is K, and mutations also exist at other amino acid sites, specifically including: (B1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (B2) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B3) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B4) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B5) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B6) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, and the 599th position mutates to K; (B7) The 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B8) The 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B9) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B10) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B11) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B12) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (B13) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B14) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 601st position mutates to R, and the 943rd position mutates to R; (B15) The 7th position is mutated to R, the 233rd position is mutated to R, the 267th position is mutated to R, the 370th position is mutated to R, the 397th position is mutated to K, the 505th position is mutated to R, the 599th position is mutated to K, and the 944th position is mutated to R; (B16) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; The mutation at amino acid position 599 of (C) is K, and mutations also exist at other amino acid sites, specifically including: (C1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C2) The 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C3) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (C4) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C5) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C6) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C7) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C8) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C9) The 7th position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C10) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 494th position mutates to K, the 599th position mutates to K, and the 943rd position mutates to R; (C11) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C12) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (C13) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C14) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; K is lysine, and R is arginine.

[0010] In a second aspect, the present invention provides a fusion protein comprising the aforementioned Cas mutant protein.

[0011] In a third aspect, the present invention provides an isolated nucleic acid molecule, said nucleic acid molecule being a nucleotide sequence encoding the above-mentioned Cas mutant protein or encoding the above-mentioned fusion protein.

[0012] In a fourth aspect, the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0013] A fifth aspect of the present invention provides a composition comprising: (A1) Protein components selected from: the Cas mutant protein described above or the fusion protein described above; (A2) Nucleic acid component, which is a guide RNA, said guide RNA being able to bind the aforementioned Cas mutant protein; The protein components and nucleic acid components combine to form a composition.

[0014] In a sixth aspect, the present invention provides an engineered host cell comprising the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, or the above-described composition.

[0015] In a seventh aspect, the present invention provides the use of the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described composition, or the above-described host cell in gene editing, gene targeting, or gene cutting, wherein the use is for purposes other than disease diagnosis and treatment; or in the preparation of reagents or kits for gene editing, gene targeting, or gene cutting.

[0016] In an eighth aspect, the present invention provides a kit for gene editing, gene targeting, or gene cutting, the kit comprising the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described composition, or the above-described host cell.

[0017] Beneficial Effects: This invention provides a Cas mutant protein with enhanced gene editing activity and its applications. Compared with existing technologies, the advantages of this invention are as follows: This invention obtains a series of amino acid sites that enhance gene editing activity through single-point mutation screening. Based on the highly active single-point mutations obtained through screening and the known Cas-SF01 mutations, mutation sites are combined on wild-type Cas12i3, and the activity differences between them and Cas-SF01 variants are evaluated. The results show that the gene editing activity of many engineered combined variants exceeds that of Cas-SF01, especially the combined mutants (S7R;D267R;K370R;E397K;E462R;S599K;K943R) with an editing activity reaching 92.5%. Therefore, this invention significantly improves the activity of Cas proteins through combined mutations and has broad application prospects. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the vector used to detect the gene editing activity of Cas12i3 and its mutants in an embodiment of the present invention.

[0019] Figure 2 To screen for single-point mutated Cas proteins with enhanced gene-editing capabilities; (A) is a schematic diagram of the EGxxFP reporter system used for screening Cas mutant proteins; a short sequence containing the target sequence 5'-atctcttagggataacaggg-3' is inserted into EGFP to disrupt its fluorescence. Targeted cleavage of the inserted sequence allows for single-strand annealing (SSA) repair between flanking repeats, thereby restoring EGFP fluorescence. This system also constitutively expresses mCherry markers. The fluorescence of EGFP is determined by... + Cells and mCherry + The editing efficiency was assessed by the ratio of cells. HEK293T cells were transfected with a plasmid encoding Cas12i3 or a variant thereof. Cells were collected 48 hours after transfection for flow cytometry analysis. The proportion of EGFP-positive cells in the mCherry cell population was calculated to obtain the relative editing activity of Cas12i3 or its variant.

[0020] Figure 3 The editing activity of the selected single-point mutants on the EGxxFP reporter system was evaluated. A total of 56 amino acid residues in Cas12i3 were screened using a protein big language model based on selected amino acids. Based on this screening, 17 novel amino acid mutants were identified with activities exceeding 1.10 times that of Cas12i3.

[0021] Figure 4To utilize the highly active single-site mutations obtained through screening and the known point mutations of Cas-SF01, mutant site combinations were performed on Cas12i3 to evaluate the activity differences between these and Cas-SF01 variants. The results showed that many engineered combinatorial variants exhibited gene-editing activity exceeding that of Cas-SF01, particularly the combinatorial mutants (S7R;D267R;K370R;E397K;E462R;S599K;K943R), which achieved an editing activity of 92.5%. Detailed Implementation

[0022] This invention provides a Cas mutant protein with enhanced editing activity and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] The following examples are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the experiments and methods described in the examples are generally performed according to conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); and the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL*. APPROACH (edited by MJ MacPherson, BD Hames and GR Taylor (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.R. Freshney (1987)).

[0024] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0025] An embodiment of the present invention provides a Cas mutant protein with enhanced editing activity, wherein the Cas mutant protein contains at least one amino acid mutation compared to the protein shown in SEQ ID No. 1; The Cas mutant protein contains any one or more of the following amino acid mutations: mutating the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, and the 788th amino acid to serine.

[0026] K is lysine, and R is arginine.

[0027] In this invention, highly active single-point mutant Cas proteins were obtained through screening, and combined with known highly active mutation sites, mutation sites were combined on Cas12i3 to obtain many combinatorial mutants with high editing activity, such as the combinatorial mutants (S7R;D267R;K370R;E397K;E462R;S599K;K943R) with editing activity as high as 92.5%.

[0028] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0029] In this invention, the biological functions of the Cas protein include, but are not limited to, activities of binding to guide RNA, endonuclease activities, and activities of binding to and cleaving target sequences at specific sites under the guidance of guide RNA, including but not limited to Cis cleavage activities and Trans cleavage activities.

[0030] In some embodiments, compared to the protein shown in SEQ ID No. 1, it contains one amino acid mutation, wherein the amino acid mutation is to mutate the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, or the 788th amino acid to serine.

[0031] In some embodiments, compared to the protein shown in SEQ ID No. 1, it contains multiple amino acid mutations, and the amino acid mutations are selected from any of the following: (A) The fifth amino acid is mutated to K, and mutations also exist at other amino acid sites; (B) The 397th amino acid is mutated to K, and mutations also exist at other amino acid sites; (C) The 599th amino acid is mutated to K, and mutations also exist at other amino acid sites; Specifically, the mutation at position 5 of (A) is K, and mutations also exist at other amino acid sites, including: (A1) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A2) The 5th position mutates to K, the 7th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A3) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A4) The 5th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A5) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A6) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 369th position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A7) The 5th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A8) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 944th position mutates to R; (A9) The 5th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (A10) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, and the 601st position mutates to R; (A11) The 5th position mutates to K, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A12) The 5th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A13) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A14) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A15) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A16) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A17) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A18) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A19) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A20) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; The mutation at amino acid position 397 in (B) is K, and mutations also exist at other amino acid sites, specifically including: (B1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (B2) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B3) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B4) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B5) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B6) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, and the 599th position mutates to K; (B7) The 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B8) The 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B9) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B10) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B11) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B12) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (B13) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B14) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 601st position mutates to R, and the 943rd position mutates to R; (B15) The 7th position is mutated to R, the 233rd position is mutated to R, the 267th position is mutated to R, the 370th position is mutated to R, the 397th position is mutated to K, the 505th position is mutated to R, the 599th position is mutated to K, and the 944th position is mutated to R; (B16) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; The mutation at amino acid position 599 of (C) is K, and mutations also exist at other amino acid sites, specifically including: (C1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C2) The 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C3) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (C4) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C5) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C6) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C7) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C8) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C9) The 7th position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C10) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 494th position mutates to K, the 599th position mutates to K, and the 943rd position mutates to R; (C11) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C12) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (C13) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C14) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; K is lysine, and R is arginine.

[0032] An embodiment of the present invention provides a fusion protein, the fusion protein comprising the above-mentioned Cas mutant protein.

[0033] In some embodiments, the fusion protein includes the Cas mutant protein as described above and other modified moieties. The modified moieties are selected from other proteins or peptides, detectable markers, or any combination thereof. Specifically, the modified moieties are selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcriptional activation domains (e.g., VP64), transcriptional repression domains (e.g., KRAB or SID domains), nuclease domains (e.g., Fok1), and domains having activities selected from: nucleotide deaminase, methyltransferase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof.

[0034] Embodiments of the present invention also provide an isolated nucleic acid molecule, wherein the nucleic acid molecule is a nucleotide sequence encoding the above-mentioned Cas mutant protein or encoding the above-mentioned fusion protein.

[0035] In some embodiments, the nucleotide sequence is codon-optimized for expression in prokaryotic cells. In one embodiment, the nucleotide sequence is codon-optimized for expression in eukaryotic cells.

[0036] In some embodiments, the cell is an animal cell, such as a mammalian cell.

[0037] In some embodiments, the cell is a human cell.

[0038] In some embodiments, the cells are plant cells, such as those found in cultivated plants (e.g., cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

[0039] In some embodiments, the nucleic acid molecule is single-stranded or double-stranded.

[0040] Embodiments of the present invention also provide a carrier comprising the above-described nucleic acid molecules.

[0041] In some embodiments, the vector further includes a regulatory element operatively linked thereto. Specifically, the regulatory element includes one or more of the following: enhancer, transposon, promoter, terminator, leader sequence, polyadenylated nucleotide sequence, and marker gene.

[0042] In some embodiments, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0043] Embodiments of the present invention also provide a CRISPR-Cas system, characterized in that the CRISPR-Cas system includes the above-mentioned Cas mutant protein and at least one guide RNA; the guide RNA is capable of binding the above-mentioned Cas mutant protein.

[0044] In some embodiments, the at least one guide RNA targets one or more target sequences in a cell. The one or more target sequences hybridize to genomic loci of a DNA molecule encoding one or more gene products and guide the Cas protein to the genomic locus of the DNA molecule of the one or more gene products. Once at the target sequence location, the Cas protein modifies, edits, or cleaves the target sequence, thereby altering or modifying the expression of the one or more gene products.

[0045] Embodiments of the present invention also provide a composition comprising: (A1) Protein components selected from: the Cas mutant protein described above or the fusion protein described above; (A2) Nucleic acid component, which is a guide RNA, said guide RNA being able to bind the aforementioned Cas mutant protein; The protein components and nucleic acid components combine to form a composition.

[0046] Embodiments of the present invention also provide an engineered host cell comprising the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described CRISPR-Cas system, or the above-described composition.

[0047] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell (such as a mammalian cell, plant cell, etc.).

[0048] The embodiments of the present invention also provide the use of the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described CRISPR-Cas system, the above-described composition, or the above-described host cell in gene editing, gene targeting, or gene cutting, wherein the use is for purposes other than disease diagnosis and treatment; or in the preparation of reagents or kits for gene editing, gene targeting, or gene cutting.

[0049] In one embodiment, the gene editing, gene targeting, or gene cleavage is performed intracellularly and / or extracellularly. Specifically, the gene editing or editing target nucleic acid includes gene modification, gene knockout, alteration of gene product expression, mutation repair, and / or polynucleotide insertion, and gene mutation.

[0050] Embodiments of the present invention also provide a kit for gene editing, gene targeting, or gene cutting, the kit comprising the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described CRISPR-Cas system, the above-described composition, or the above-described host cell.

[0051] Embodiments of the present invention also provide the use of the above-described Cas mutant protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described vector, the above-described CRISPR-Cas system, the above-described composition, the above-described host cell, or the above-described kit in the preparation of formulations, wherein the formulations are used in any one of (B1)-(B4): (B1) In vitro gene or genome editing; (B2) Detection of isolated single-stranded DNA; (B3) Editing target sequences in target loci to modify organisms; (B4) Treating conditions caused by defects in target sequences at target loci.

[0052] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures described herein, including those related to molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional procedures widely used in their respective fields.

[0053] In this invention, "Cas mutant protein" can also be referred to as mutated Cas protein or Cas protein variant.

[0054] In this invention, "Cas12i3 single-point mutant", "Cas12i3 double-point mutant", "Cas12i3 triple-point mutant" and "Cas12i3 multi-point mutant" are all Cas mutant proteins.

[0055] In this invention, "combination mutant" can refer to "Cas12i3 two-point mutant", "Cas12i3 three-point mutant" or "Cas12i3 multi-point mutant".

[0056] The term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, E5K means that E at position 5 is mutated to K. When multiple amino acid sites are mutated simultaneously, it can be expressed in a form like E5K;S7K. For example, E5K;S7K means that E at position 5 is mutated to K and S at position 7 is mutated to K.

[0057] The embodiments of the present invention will now be described in detail with reference to examples.

[0058] Example 1 This embodiment constructs a Cas12i3 single-point mutant through single-point mutation, expresses the Cas12i3 single-point mutant in cells, and uses the EGxxFP reporter system to quantitatively compare the effects of different single-point mutations on gene editing activity, thereby screening out key mutation sites with improved editing performance. Details are as follows: 1. Single point mutation For the known Cas protein (Cas12f.4 in CN111757889B, referred to as Cas12i3 in this embodiment), the applicant used bioinformatics to predict key amino acid sites that may affect its biological function, and then performed point mutations on these amino acid sites to obtain Cas12i3 single-point mutants. Specifically, the Cas12i3 coding sequence was codon-optimized (human) and synthesized. The amino acid sequence of wild-type Cas12i3 is shown in SEQ ID No. 1, and its nucleic acid sequence is shown in SEQ ID No. 2. Bioinformatics methods were used to predict amino acid residues that may be involved in recognition or cleavage during the interaction between Cas12i3 and target DNA, and corresponding point mutations were designed for these residues to obtain different Cas12i3 single-point mutant coding sequences.

[0059] 2. Construct plasmid vectors encoding Cas12i3 or its single-point mutants: The carrier skeleton used in this embodiment is as follows: Figure 1 As shown, the Cas12i3 coding sequence or the Cas12i3 single-point mutant coding sequence is constructed into a mammalian expression vector to obtain plasmid vectors that can express Cas12i3 in cells and plasmid vectors that express the Cas12i3 single-point mutant. This can be done using methods commonly used in the art for constructing plasmid vectors. The specific method in this embodiment is as follows: the coding sequence of the Cas12i3 single-point mutant is synthesized as a fragment (commissioned by GenScript), and then the synthesized fragment is assembled into a mammalian expression vector using double enzyme digestion and homologous recombination; wherein the enzyme digestion sites used in this embodiment are XbaI and PstI.

[0060] 3. Cell culture, transfection, and flow cytometry analysis: like Figure 2 As shown, this invention uses the EGxxFP reporter system to evaluate the gene editing activity of Cas12i3 and Cas12i3 single-point mutants. By inserting a target sequence into the EGFP coding sequence to disrupt its fluorescent expression, when Cas12i3 cleaves the inserted sequence under crRNA guidance, it triggers single-stranded annealing (SSA) repair between flanking repeats, thereby restoring the EGFP fluorescent signal. This system also constitutively expresses mCherry as a transfection marker, and the gene editing activity is evaluated by comparing EGFP levels. +Cells in mCherry + The proportion of different Cas12i3 single-point mutants in the cell population was used to evaluate their relative editing activity. The specific experimental steps are as follows: HEK293T cells were cultured in high-glucose DMEM medium supplemented with 10% (v / v) fetal bovine serum (Gibco, 10099141) and 1% penicillin / streptomycin (Gibco, 15140122) at 37°C and 5% CO2.

[0061] HEK293T cells were maintained under standard culture conditions and seeded in 12-well plates at approximately 80% confluence before transfection. For each transfection, 1 μg of plasmid DNA was diluted in 100 μL of Opti-MEM (Gibco, 51985091), followed by the addition of 3 μL of polyethyleneimine (PEI, Yeasen, 40816ES02). The DNA / PEI complex was incubated at room temperature for 20 minutes before being added to the cells. For EGxxFP reporter gene assays, cells were harvested 48 hours post-transfection and analyzed by flow cytometry.

[0062] After removing the culture medium, cells transfected in each well were digested with 300 μL of 0.25% trypsin (Gibco, 25200-056). Digestion was then stopped with 300 μL of culture medium, and the cells were centrifuged at 1000 rpm for 3 minutes. The cells were then resuspended in 500 μL of culture medium. Cells were pipetteed into 5 mL round-bottom polystyrene tubes (Corning, 352235) with cell filter caps. Flow cytometry data were collected using CytoFLEX (Beckman Coulter Life Sciences) and analyzed using CyExpertversion 2.5.0.77 software.

[0063] Inactive and dual-cell cells were removed via a scattering gate. For positive assays, the gate was set based on cells co-transfected with mCherry and GFP signals. mCherry fluorescence was detected using Y610 (561 / 610 nm). GFP fluorescence was detected using B525 (488 / 525 nm). Approximately 30,000 cells were collected per sample (after scattering gate). Editing efficiency was calculated as GFP. + Cell count divided by mCherry + Cell count.

[0064] 4. Results: like Figure 3 As shown in the figure, a single-point mutation was performed based on SEQ ID No. 1 in this embodiment. Based on the results of this figure, mutation sites with significantly enhanced editing activity were further screened. The obtained mutation sites are shown in Table 1: Table 1. Relative editing activity of Cas12i3 single-point mutants

[0065] Based on the aforementioned amino acid mutation sites, wild-type Cas12i3 proteins (hereinafter referred to as I3_WT and I3T2-WT), as well as Cas proteins with single-point mutations at different amino acid sites based on the wild-type Cas12i3 protein, were obtained. The single-point mutants include: 5K, 7K, 396K, 397K, 462R, 494K, 599K, and 788S. Relative to the Cas12i3 amino acid sequence shown in SEQ ID No. 1, these mutants have K, R, or S substitution mutations at amino acids 5, 7, 396, 397, 462, 494, 599, and 788, starting from the N-terminus.

[0066] In the following embodiments, highly active single-point mutations and known point mutations obtained through the above screening were combined on wild-type Cas12i3 to determine which combination of mutants was more effective.

[0067] Example 2 This embodiment constructs a Cas12i3 double-point mutant (double mutant) based on the mutation sites screened in Example 1, and verifies the effect of the double mutant on gene editing activity, as detailed below: 1. Construct plasmid vectors encoding Cas12i3 or double mutations: Same as in Example 1.

[0068] 2. Cell culture, transfection, and flow cytometry analysis: Same as in Example 1.

[0069] 3. Results: As shown in Table 2, the double mutation constructed in this embodiment is a process mutation, and subsequent experiments will further combine mutations based on this.

[0070] Table 2. Relative editing activity of Cas12i3 two-point mutants

[0071] Example 3 This embodiment constructs a Cas12i3 three-point mutant (triple mutant) or a Cas12i3 four-point mutant (quadruple mutant) based on the mutation sites screened in Example 1, and verifies the effect of the three-point or four-point mutant on gene editing activity, as detailed below: 1. Construct plasmid vectors encoding Cas12i3, with three or four protrusions: Same as in Example 1.

[0072] 2. Cell culture, transfection, and flow cytometry analysis: Same as in Example 1.

[0073] 3. Results: As shown in Table 3, the three or four mutations constructed in this embodiment are also process mutations. Subsequent experiments will further combine mutations based on this.

[0074] Table 3. Relative editing activity of Cas12i3 three-point mutants or Cas12i3 four-point mutants

[0075] Example 4 In this embodiment, a Cas12i3 multi-point mutant (multi-mutant) was constructed based on the mutation sites obtained in Example 1, wherein the mutation sites of the multi-mutant are greater than 4; and the effect of the multi-mutant on gene editing activity was verified. In addition, a Cas protein BC26312 with amino acid mutations is disclosed in patent CN116004573B, which is referred to as Cas-SF01 in this embodiment. The multi-mutant obtained in this embodiment is compared with Cas-SF01, and a series of Cas proteins with higher editing activity than Cas-SF01 are obtained.

[0076] Specifically as follows: 1. Constructing a Cas12i3 or multi-protrusion vector: Same as in Example 1.

[0077] 2. Cell culture, transfection, and flow cytometry analysis: Same as in Example 1.

[0078] 3. Results: This embodiment yielded a series of combined mutants. Further screening of these combined mutants revealed combined mutants with high editing activity (as shown in Table 4 and...). Figure 4 As shown in Table 4, the editing activities of the combined mutants provided by this invention are all higher than those of Cas-SF01 (SF01). The combined mutants are sorted from low to high according to the expression level of Indel (%).

[0079] Table 4. Relative editing activity of Cas12i3 multi-point mutants

[0080] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A Cas mutant protein with enhanced editing activity, characterized in that, The Cas mutant protein contains at least one amino acid mutation compared to the protein shown in SEQ ID No. 1; The Cas mutant protein contains any one or more of the following amino acid mutations: mutating the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, and the 788th amino acid to serine.

2. The Cas mutant protein according to claim 1, characterized in that, Compared with the protein shown in SEQ ID No. 1, it contains one amino acid mutation, wherein the amino acid mutation is to change the 5th amino acid of SEQ ID No. 1 to lysine, the 7th amino acid to lysine, the 396th amino acid to lysine, the 397th amino acid to lysine, the 462nd amino acid to arginine, the 494th amino acid to lysine, the 599th amino acid to lysine, or the 788th amino acid to serine.

3. The Cas mutant protein according to claim 1, characterized in that, Compared to the protein shown in SEQ ID No. 1, it contains multiple amino acid mutations, and said amino acid mutations are selected from any of the following: (A) The fifth amino acid is mutated to K, and mutations also exist at other amino acid sites; (B) The 397th amino acid is mutated to K, and mutations also exist at other amino acid sites; (C) The 599th amino acid is mutated to K, and mutations also exist at other amino acid sites; Specifically, the mutation at position 5 of (A) is K, and mutations also exist at other amino acid sites, including: (A1) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A2) The 5th position mutates to K, the 7th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A3) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A4) The 5th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A5) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A6) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 369th position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A7) The 5th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A8) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 944th position mutates to R; (A9) The 5th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (A10) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, and the 601st position mutates to R; (A11) The 5th position mutates to K, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A12) The 5th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A13) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A14) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; (A15) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A16) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A17) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A18) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (A19) The 5th position mutates to K, the 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 601st position mutates to R, and the 944th position mutates to R; (A20) The 5th position mutates to K, the 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 433rd position mutates to R, the 505th position mutates to R, and the 601st position mutates to R; The mutation at amino acid position 397 in (B) is K, and mutations also exist at other amino acid sites, specifically including: (B1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, and the 599th position mutates to K; (B2) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B3) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B4) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B5) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (B6) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, and the 599th position mutates to K; (B7) The 7th position mutates to K, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B8) The 7th position mutates to K, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B9) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B10) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B11) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (B12) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (B13) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (B14) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 601st position mutates to R, and the 943rd position mutates to R; (B15) The 7th position is mutated to R, the 233rd position is mutated to R, the 267th position is mutated to R, the 370th position is mutated to R, the 397th position is mutated to K, the 505th position is mutated to R, the 599th position is mutated to K, and the 944th position is mutated to R; (B16) The 7th position mutates to K, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; The mutation at amino acid position 599 of (C) is K, and mutations also exist at other amino acid sites, specifically including: (C1) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C2) The 7th position mutates to K, the 267th position mutates to R, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C3) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 494th position mutates to K, the 599th position mutates to K, and the 944th position mutates to R; (C4) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C5) The 7th position mutates to K, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C6) The 7th position mutates to R, the 267th position mutates to R, the 397th position mutates to K, the 433rd position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C7) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 397th position mutates to K, the 505th position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C8) The 7th position mutates to R, the 233rd position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C9) The 7th position mutates to R, the 267th position mutates to R, the 328th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C10) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 494th position mutates to K, the 599th position mutates to K, and the 943rd position mutates to R; (C11) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 505th position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; (C12) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, the 601st position mutates to R, and the 943rd position mutates to R; (C13) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 944th position mutates to R; (C14) The 7th position mutates to R, the 267th position mutates to R, the 370th position mutates to R, the 462nd position mutates to R, the 599th position mutates to K, and the 943rd position mutates to R; K is lysine, and R is arginine.

4. A fusion protein comprising the Cas mutant protein according to any one of claims 1-3.

5. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleotide sequence encoding the Cas mutant protein of any one of claims 1-3 or the fusion protein of claim 4.

6. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 5.

7. A composition, characterized in that, The composition comprises: (A1) A protein component selected from: the Cas mutant protein according to any one of claims 1-3 or the fusion protein according to claim 4; (A2) A nucleic acid component, which is a guide RNA, said guide RNA being capable of binding to the Cas mutant protein according to any one of claims 1-3; The protein components and nucleic acid components combine to form a composition.

8. An engineered host cell, characterized in that, The host cell comprises the Cas mutant protein of any one of claims 1-3, the fusion protein of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, or the composition of claim 7.

9. The use of the Cas mutant protein of any one of claims 1-3, the fusion protein of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, the composition of claim 7, or the host cell of claim 8 in gene editing, gene targeting, or gene cutting, wherein the use is for purposes other than disease diagnosis and treatment; or in the preparation of reagents or kits for gene editing, gene targeting, or gene cutting.

10. A kit for gene editing, gene targeting, or gene cutting, the kit comprising the Cas mutant protein of any one of claims 1-3, the fusion protein of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, the composition of claim 7, or the host cell of claim 8.

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