A polypeptide carrier that mediates delivery of crisper-cas gene editing tools

Abstract

The present application relates to the technical field of gene editing, in particular to a CRISPR-Cas gene editing tool delivery vector. The present application provides a polypeptide and its application, the polypeptide can assist the delivery of Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in the CRISPR-Cas gene editing system into the target cells, the damage caused to the cells is reduced, and the inherent characteristics and state of the cells are almost not affected; the operation process is simple and rapid; and the polypeptide has the advantages of low cost.

Description

[0001] Priority information This invention claims priority and benefit to Chinese Patent Application No. 202510532650.9, filed with the China National Intellectual Property Administration on April 25, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of gene editing technology, and more specifically to a polypeptide vector that mediates the delivery of CRISPR-Cas gene editing tools. Background Technology

[0003] CRISPR-Cas systems, as powerful gene-editing tools, have shown broad application prospects in gene function research and gene therapy for diseases. Current methods for delivering CRISPR-Cas systems mainly include viral vectors, non-viral vectors, physical methods, and chemical methods. Viral vectors, such as adeno-associated virus (AAV), lentivirus, and adenovirus, can efficiently infect and target specific cell types. However, AAV has a limited packaging capacity (~4.7 kb), making it difficult to accommodate larger CRISPR-Cas systems (such as SpCas9), and may trigger a host immune response, affecting delivery efficiency, and carries the risk of insertional mutations. Non-viral vectors, such as liposomes, nanoparticles, and polymer vectors, have the advantages of high safety and no risk of insertional mutations, but their delivery efficiency is low. Compared to viral vectors, non-viral vectors generally have lower delivery efficiency and poor targeting and stability. Commonly used physical methods, such as electroporation, microinjection, and gene guns, can directly and efficiently deliver CRISPR-Cas systems in vitro, but they can damage cells, affecting cell viability and are not suitable for in vivo applications, making them difficult to use in living tissues or animal models. Therefore, existing technologies for delivering CRISPR-Cas systems to target cells all have certain drawbacks, such as limited viral vector packaging capacity, high cytotoxicity, high immunogenicity, and low primary cell delivery efficiency, which seriously restrict the promotion of CRISPR-Cas systems in clinical applications such as gene therapy.

[0004] To overcome the aforementioned problems, researchers have proposed a novel method for delivering CRISPR-Cas systems using peptides as vectors. Compared to viral vectors, peptide vectors offer advantages such as better biocompatibility, lower cytotoxicity, and simpler preparation, potentially improving the delivery safety and efficiency of CRISPR-Cas systems. However, existing peptide vectors still have limitations in delivering CRISPR-Cas systems, such as unstable binding between the vector and the CRISPR-Cas system and low intracellular release efficiency, which affect the effectiveness of gene editing. Therefore, there is an urgent need to develop a novel peptide vector CRISPR-Cas delivery system to further improve the efficiency and safety of CRISPR-Cas systems in gene editing, providing strong support for gene therapy, disease model construction, and other applications. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a polypeptide and its application, said polypeptide being able to assist in the delivery of the Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in a CRISPR-Cas gene editing system into target cells, reducing damage to cells and having almost no impact on the inherent characteristics and state of the cells; the operation process is simple and rapid; and it also has the advantage of low cost.

[0006] Therefore, a first aspect of the present invention provides a polypeptide. According to an embodiment of the present invention, the polypeptide comprises a cell-penetrating peptide and an antimicrobial peptide (AMP) amino acid sequence, wherein the C-terminus of the cell-penetrating peptide is linked to the N-terminus of the antimicrobial peptide amino acid sequence; or, The C-terminus of the amino acid sequence of the antimicrobial peptide is linked to the N-terminus of the cell-penetrating peptide. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1-19, or is a derived sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-19.

[0007] The inventors have discovered a polypeptide comprising a cell-penetrating peptide and an antimicrobial peptide amino acid sequence that can assist the delivery of the Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in the CRISPR-Cas gene editing system into target cells, reducing cell damage and having almost no impact on the cell's inherent properties and state.

[0008] In some embodiments of the present invention, the cell-penetrating peptide includes at least one selected from TAT, (Arg)9, Penetratin, GALA, and LAH4.

[0009] When the cell-penetrating peptide is at least one of TAT, (Arg)9, Penetratin, GALA, and LAH4, the polypeptide formed by it and the antimicrobial peptide can assist the delivery of the Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in the CRISPR-Cas gene editing system into the target cell, reducing the damage to the cell and having almost no effect on the cell's inherent properties and state.

[0010] In some embodiments of the present invention, the cell-penetrating peptide is TAT ​​or (Arg)9.

[0011] In some embodiments of the present invention, the amino acid sequence of the TAT is as shown in SEQ ID NO: 24 and SEQ ID NO: 25, or is a derived sequence having the same function as the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 25.

[0012] In some embodiments of the present invention, the amino acid sequence of (Arg)9 is as shown in SEQ ID NO: 30, or is a derived sequence having the same function as the amino acid sequence shown in SEQ ID NO: 30.

[0013] In some embodiments of the present invention, at least one amino acid in the amino acid sequence of the cell-penetrating peptide and / or antimicrobial peptide is a D-type amino acid.

[0014] In some embodiments of the present invention, the amino acid sequences of the cell-penetrating peptide and / or antimicrobial peptide are unmodified or chemically modified.

[0015] In some embodiments of the present invention, the chemical modification includes amidation or sulfation of C-terminal amino acids; acetylation or fatty acidation of N-terminal amino acids; glycosylation of at least one of the intermediate residues serine, tyrosine, tryptophan, and asparagine; phosphorylation of at least one of the intermediate residues serine, tyrosine, and tryptophan; cyclization; and polyethylene glycol modification.

[0016] The inventors discovered that by chemically modifying the amino acid sequences of the cell-penetrating peptide and / or antimicrobial peptide as described above, such peptides can assist the delivery of the Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in the CRISPR-Cas gene editing system into the target cell, reducing damage to the cell and having almost no impact on the cell's inherent properties and state.

[0017] In some embodiments of the present invention, in the polypeptide, the C-terminus of the amino acid sequence of TAT or (Arg)9 is directly linked to the N-terminus of the amino acid sequence of the antimicrobial peptide; or, The C-terminus of the antimicrobial peptide amino acid sequence is directly linked to the N-terminus of the amino acid sequence of TAT or (Arg)9.

[0018] In some embodiments of the present invention, the amino acid sequence of TAT is as shown in SEQ ID NO: 24 or SEQ ID NO: 25; and the amino acid sequence of (Arg)9 is as shown in SEQ ID NO: 30.

[0019] In some embodiments of the present invention, the amino acid sequences of the antimicrobial peptide are as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO: 18.

[0020] In some embodiments of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. A second aspect of the present invention provides the use of the polypeptide described in the first aspect in improving the gene editing efficiency of the CRISPR / Cas system.

[0021] In some embodiments of the present invention, the Cas protein involved in the CRISPR / Cas system is a CRISPR system effector protein.

[0022] In some embodiments of the present invention, the CRISPR system effector protein includes one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

[0023] The third aspect of this invention provides the use of the polypeptide described in the first aspect in the preparation of reagents or kits for improving the gene editing efficiency of CRISPR / Cas systems.

[0024] In some embodiments of the present invention, the Cas protein involved in the CRISPR / Cas system is a CRISPR system effector protein.

[0025] In some embodiments of the present invention, the CRISPR system effector protein includes one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

[0026] A fourth aspect of this invention provides a reagent or kit for improving the gene editing efficiency of CRISPR / Cas systems. In some embodiments of this invention, the reagent or kit comprises the polypeptide described in the first aspect.

[0027] A fifth aspect of this invention provides a method for improving the gene editing efficiency of a CRISPR / Cas system. In some embodiments of this invention, the method includes: The polypeptide described in the first aspect is mixed with the delivery cargo and introduced into the target cells. The delivered goods include: A complex of sgRNA or crRNA targeting the target gene and CRISPR system effector proteins, or the CRISPR system effector proteins themselves.

[0028] In some embodiments of the present invention, the CRISPR system effector protein includes one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

[0029] A sixth aspect of the present invention provides a fusion protein. In some embodiments of the present invention, the fusion protein comprises the amino acid sequence of at least one polypeptide described in the first aspect.

[0030] In some embodiments of the present invention, the C-terminus of the polypeptide is linked to the N-terminus of a CRISPR system effector protein; or, The N-terminus of the polypeptide is linked to the C-terminus of a CRISPR system effector protein.

[0031] In some embodiments of the present invention, the CRISPR system effector protein includes one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

[0032] A seventh aspect of the present invention provides a nucleic acid molecule. In some embodiments of the present invention, the nucleic acid molecule encodes the fusion protein described in the sixth aspect.

[0033] An eighth aspect of the present invention provides an expression vector. In some embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the seventh aspect.

[0034] A ninth aspect of the present invention provides a recombinant virus. In some embodiments of the present invention, the recombinant virus comprises the expression vector described in the eighth aspect, and the recombinant virus is capable of infecting target cells.

[0035] Compared to existing technologies, the peptides provided by this invention can achieve high editing effects in most cell types (such as EL4, THP-1, Jurkat), and even in human primary T cells with higher operational complexity, using fewer cells; reduce damage to cells and hardly affect the inherent characteristics and state of cells; the operation process is simple and fast; and it also has the advantage of lower cost.

[0036] The peptides of this invention can enhance the efficiency of gene editing in cell lines and primary cells with high efficiency, low toxicity, and low perturbation. This invention also provides a method for improving the gene editing efficiency of the CRISPR / Cas system, which significantly improves the simplicity of operation and greatly shortens the experimental cycle compared to existing methods. Furthermore, this invention establishes the principles for constructing antimicrobial peptide vector sequences.

[0037] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 A schematic diagram of the TAT-AMP delivery Cas9 editing system in Example 2 is shown. Figure 2 The gene editing effects of TAT-HA2 and TAT-AMP in EL4 mCherry cells were shown in Example 2; Figure 3 The gene editing effects of TAT-HA2 and TAT-AMP in THP-1 mCherry cells were shown in Example 2; Figure 4 A schematic diagram of the TAT-AMP delivery Cas12a editing system in Embodiment 2 is shown; Figure 5 This demonstrates the delivery of TAT-AMP to RNP-knockout Jurkat cells in Example 2. B2M The process of targeting genes; Figure 6 The effect of RNP delivery using TAT-AMP on B2M protein levels in the Jurkat cell line was shown by flow cytometry in Example 2. Figure 7 The editing effects of the peptides, TAT-HA2 and TAT-AMP at a final concentration of 10 μM in Example 2 on human primary T cells are shown. Figure 8The editing effects of the peptides, TAT-HA2 and TAT-AMP at a final concentration of 25 μM in Example 2 on human primary T cells are shown. Figure 9 The results of the toxicity test on human primary T cells at a final concentration of 25 μM TAT-HA2 and TAT-AMP in Example 2 are shown. Figure 10 The results of flow cytometry analysis of surface B2M protein levels were shown on day 10 after B2M gene knockout in Example 2. Figure 11 The effect of RNP delivery with a modified 25 μM final concentration of (Arg)9-AMP on B2M protein levels in the Jurkat cell line was shown by flow cytometry in Example 2. Figure 12 The gene editing effect of the modified 25 μM final concentration of (Arg)9-AMP in EL4 mCherry cells is shown in Example 2; Figure 13 The editing effect of TAT-AMP, after C-terminal acetylation modification, on Jurkat cells is shown at a final concentration of 25 μM of the peptide in Example 2. Figure 14 The editing effect of TAT-AMP, after C-terminal acetylation modification, on EL4mCherry cells at a final concentration of 25 μM of the peptide in Example 2 is shown. Figure 15 The editing effect of fatty acid-modified TAT-AMP on Jurkat cells is shown at a final concentration of 25 μM of the peptide in Example 2. Figure 16 The results show the editing effect of the 25 μM final concentration of the peptide from Example 2, after fatty acid modification, on EL4mCherry cells. Detailed Implementation

[0039] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0040] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0041] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0042] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0044] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0045] This invention first uses bioinformatics to predict the sequence of an antimicrobial peptide (AMP) with endosome escape capability, and synthesizes a fusion polypeptide (TAT-AMP) of the cell-penetrating peptide TAT and the antimicrobial peptide (AMP), giving it dual functions of cell penetration and endosome escape: it can efficiently and relatively gently deliver the CRISPR-Cas system into cells and promote the endosome escape of the CRISPR-Cas system in the cytoplasm, thereby improving its nuclear entry capability and gene editing. Secondly, the delivery effect of the fusion polypeptide (TAT-AMP) on the CRISPR-Cas system in primary cells and cell lines is tested. Finally, fusion peptide vector sequences capable of delivering the CRISPR-Cas system are screened. Thus, the polypeptide sequence of this invention is obtained.

[0046] According to a specific embodiment of the present invention, the present invention provides a polypeptide comprising a cell-penetrating peptide and an AMP amino acid sequence, wherein the C-terminus of the cell-penetrating peptide is linked to the N-terminus of the AMP amino acid sequence; or, The C-terminus of the AMP amino acid sequence is linked to the N-terminus of the cell-penetrating peptide. The AMP amino acid sequence is as shown in SEQ ID NO: 1-19, or is a derived sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-19.

[0047] It should be noted that the cell-penetrating peptide and the AMP amino acid sequence in the polypeptide of the present invention can be directly linked, or they can be linked by a linker, provided that the linker does not affect the function of the cell-penetrating peptide and the AMP amino acid sequence itself.

[0048] Among the polypeptides described in this invention, cell-penetrating peptides include, but are not limited to, TAT, (Arg)9, Penetratin, GALA, LAH4, etc.

[0049] According to a preferred embodiment of the present invention, the cell-penetrating peptide is TAT. It should be noted that the TAT amino acid sequence in the polypeptide of the present invention can be the amino acid sequence shown in SEQ ID NO: 24 and SEQ ID NO: 25, or a derived sequence having the same function as the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 25, both of which are covered within the protection scope of the polypeptide of the present invention. Specifically, the amino acid sequence shown in SEQ ID NO: 24 is RRRQRRKKRG, the amino acid sequence shown in SEQ ID NO: 25 is GRKKRRQRRR, and SEQ ID NO: 25 is the reverse sequence of the amino acid sequence shown in SEQ ID NO: 24.

[0050] Derivative sequences having the same function as the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 25 can be, for example, GRKKRRQRRRPQ (SEQ ID NO: 26), GRKKRRQRRRPPQ (SEQ ID NO: 27), YGRKKRRQRRR (SEQ ID NO: 28), AYGRKKRRQRRR (SEQ ID NO: 29), etc.

[0051] According to a specific embodiment of the present invention, at least one amino acid in the cell-penetrating peptide and / or AMP amino acid sequence is a D-type amino acid.

[0052] In some embodiments of the present invention, the cell-penetrating peptide and / or AMP amino acid sequence are either unmodified or chemically modified. There are no particular limitations on the selection of chemical groups; the chemically modified peptide functions similarly to the unmodified peptide, and all chemical groups meeting this requirement are covered within the scope of chemical modification in this invention.

[0053] In some embodiments of the present invention, the chemical modification includes amidation or sulfation of C-terminal amino acids; acetylation or fatty acidation of N-terminal amino acids; glycosylation of at least one of the intermediate residues serine, tyrosine, tryptophan, and asparagine; phosphorylation of at least one of the intermediate residues serine, tyrosine, and tryptophan; cyclization; and polyethylene glycol modification.

[0054] According to a specific embodiment of the present invention, the chemically modified amino acid sequence is as follows: RRRQRRKKRGARHTALVPLWQCQIR-NH2 (SEQ ID NO: 34) stearyl-RRRQRRKKRGARHTALVPLWQCQIR-NH2 (SEQ ID NO: 35) Myristic acid-RRRQRRKKRGARHTALVPLWQCQIR-NH2 (SEQ ID NO: 36).

[0055] It should be noted that the TAT amino acid sequence in the polypeptide of this invention can be replaced by any cell-penetrating polypeptide (CPP) sequence, such as... (Arg)9: RRRRRRRRR (SEQ ID NO: 30); Penetratin: RQIKIWFQNRRMKWKKGG (SEQ ID NO: 31); GALA: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 32); LAH4: KKALLALALHHLAHLALHLALALKKA (SEQ ID NO: 33), etc.

[0056] It should be noted that the AMP amino acid sequence in the polypeptide of the present invention can be replaced by any known AMP sequence other than SEQ ID NO: 1-19.

[0057] The inventors discovered that in the peptide, whether the TAT or (Arg)9 amino acid sequence is at the N-terminus of the peptide, or the AMP amino acid sequence is at the N-terminus of the peptide, this form of peptide can assist the delivery of the Cas protein and its ribonucleoprotein complex (RNP) with sgRNA or crRNA in the CRISPR-Cas gene editing system into the target cell, further improving gene editing efficiency.

[0058] According to a specific embodiment of the present invention, the amino acid sequence of the polypeptide after replacing the N-terminal TAT with (Arg)9 is as follows: RRRRRRRRRFLAKGKKSLKTWGAPKDIIIIGNAM (SEQ ID NO: 37) RRRRRRRRRARHTALVPLWQCQIR (SEQ ID NO: 38).

[0059] According to a specific embodiment of the present invention, in the polypeptide, the C-terminus of the TAT or (Arg)9 amino acid sequence is directly linked to the N-terminus of the AMP amino acid sequence; or, The C-terminus of the AMP amino acid sequence is directly linked to the N-terminus of the TAT or (Arg)9 amino acid sequence.

[0060] According to a specific embodiment of the present invention, the TAT amino acid sequence is as shown in SEQ ID NO: 24 or SEQ ID NO: 25. Preferably, the TAT amino acid sequence is as shown in SEQ ID NO: 24.

[0061] According to a preferred embodiment of the present invention, the AMP amino acid sequence is as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO: 18.

[0062] According to a specific embodiment of the present invention, the TAT amino acid sequence is shown in SEQ ID NO: 24.

[0063] According to a specific embodiment of the present invention, the amino acid sequence of (Arg)9 is RRRRRRRRR (SEQ ID NO: 30).

[0064] According to a specific embodiment of the present invention, the present invention provides a polypeptide composed of a TAT amino acid sequence and an AMP amino acid sequence, wherein the C-terminus of the TAT amino acid sequence is directly linked to the N-terminus of the AMP amino acid sequence, the TAT amino acid sequence is shown in SEQ ID NO: 24, and the AMP amino acid sequence is shown in SEQ ID NO: 1-19. Preferably, the AMP amino acid sequence is shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO: 18.

[0065] According to a specific embodiment of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.

[0066] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned peptide in improving the gene editing efficiency of the CRISPR / Cas system.

[0067] It should be noted that the Cas proteins involved in the CRISPR / Cas system include, but are not limited to, CRISPR system effector proteins. According to a specific embodiment of the present invention, the CRISPR system effector proteins include one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

[0068] According to a preferred embodiment of the present invention, the class II CRISPR system effector proteins are Cas12a and Cas9.

[0069] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned polypeptide in the preparation of reagents or kits for improving the gene editing efficiency of CRISPR / Cas systems.

[0070] According to a specific embodiment of the present invention, the present invention provides a reagent or kit for improving the gene editing efficiency of the CRISPR / Cas system, the reagent or kit comprising the aforementioned polypeptide.

[0071] It should be noted that the reagent may also contain other components, and the kit may also contain other reagents, all of which are covered within the scope of protection of this invention.

[0072] According to a specific embodiment of the present invention, the present invention provides a method for improving the gene editing efficiency of the CRISPR / Cas system, the method comprising: The aforementioned peptides and delivery cargo are mixed and introduced into the target cells. The delivered goods include: A complex of sgRNA or crRNA targeting the target gene and CRISPR system effector proteins, or the CRISPR system effector proteins themselves.

[0073] It should be noted that the target cells can be of various cell types, including but not limited to EL4, THP-1, and Jurkat cell types. In particular, the method provided by this invention can also improve the gene editing efficiency of the CRISPR / Cas system for human primary T cells, which have higher operational complexity.

[0074] It should be noted that the CRISPR system effector proteins include, but are not limited to, one or more of Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5 and Fokl, or other CRISPR system effector proteins, all of which are covered within the scope of protection of this invention.

[0075] According to a more specific embodiment of the present invention, when the CRISPR system effector protein is Cas9 protein, the method for improving the gene editing efficiency of the CRISPR / Cas system includes the following steps: (1) Introduce a vector that expresses sgRNA designed to target the gene into the target cell to obtain the modified target cell; (2) The modified target cells are mixed with Cas9 protein and the polypeptide of the present invention (TAT-AMP or (Arg)9-AMP) and incubated to obtain gene-edited target cells, and the gene editing efficiency of the target cells is significantly improved compared with the gene editing method without TAT-AMP.

[0076] It should be noted that during mixed incubation, the peptide TAT-AMP or (Arg)9-AMP can be mixed with Cas9 first, or Cas9 can be mixed with the cells first, and then the peptide can be added. The target cells can be various cell types, including but not limited to EL4, THP-1, and other cell types.

[0077] There are no particular restrictions on the culture conditions, concentrations of Cas9 protein, peptide, and cell concentration during incubation. Preferably, the final concentration of the peptide TAT-AMP or (Arg)9-AMP is 25-75 μM, and the final concentration of the Cas9 protein is 0.5 μM. The incubation temperature can be, for example, 37°C, and the incubation time can be adjusted as needed, for example, to 30 minutes.

[0078] According to a more specific embodiment of the present invention, when the effector protein of the class II CRISPR system is Cas12a protein, the method for improving the gene editing efficiency of the CRISPR / Cas system includes the following steps: (1) Prepare a complex RNP containing Cas12a protein and crRNA in vitro, wherein the crRNA is designed to target the gene; the molar ratio of Cas12a protein to crRNA is 1.25:1.

[0079] (2) The complex is mixed with the polypeptide (TAT-AMP or (Arg)9-AMP) and the target cells and incubated to obtain gene-edited target cells, and the gene editing efficiency in the target cells is significantly improved compared with the gene editing method without TAT-AMP or (Arg)9-AMP.

[0080] It should be noted that during mixed incubation, the polypeptide (TAT-AMP or (Arg)9-AMP) and Cas12aRNP can be mixed first. The target cells can be various cell types, including but not limited to Jurkat, THP-1, and human primary T cells.

[0081] There are no particular restrictions on the culture conditions during incubation, as well as the concentrations of Cas12a protein, peptide, and cell concentration. Preferably, the final concentration of the peptide TAT-AMP or (Arg)9-AMP is 10-75 μM, and the final concentration of the Cas12a protein is 6.25 μM. The incubation temperature can be, for example, 37°C, and the incubation time can be adjusted as needed, for example, 30 minutes.

[0082] According to a specific embodiment of the present invention, the present invention provides a fusion protein comprising the amino acid sequence of at least one of the aforementioned polypeptides.

[0083] According to a specific embodiment of the present invention, the C-terminus of the polypeptide is linked to the N-terminus of a CRISPR system effector protein; or, The N-terminus of the polypeptide is linked to the C-terminus of a CRISPR system effector protein.

[0084] The polypeptide sequence can be fused with CRISPR system effector proteins for expression to improve gene editing efficiency.

[0085] According to a specific embodiment of the present invention, the CRISPR system effector protein includes one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5 and Fokl.

[0086] According to a specific embodiment of the present invention, the present invention provides a nucleic acid molecule that encodes the aforementioned fusion protein.

[0087] According to a specific embodiment of the present invention, the present invention provides an expression vector comprising the aforementioned nucleic acid molecules.

[0088] According to a specific embodiment of the present invention, the present invention provides a recombinant virus comprising the aforementioned expression vector, wherein the recombinant virus is capable of infecting target cells.

[0089] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0090] Example 1: Screening of AMP The antimicrobial peptides screened in this invention were derived from three papers: *Discovery of antimicrobial peptides in the global microbiome with machine learning*, *Mining human microbiomes reveals an untapped source of peptide antibiotics*, and *Global marine microbial diversity and its potential in bioprospecting*. A total of 299 antimicrobial peptide sequences were collected. The peptide sequence length was selected to be 25-30 amino acid residues, partly to reduce the cost of peptide synthesis, and partly because sequences that are too short are not conducive to maintaining a stable structure and penetrating cells. 133 sequences with a length of 25 amino acid residues or less, and 136 sequences with a length between 26 and 35 amino acid residues, were selected for further screening.

[0091] AMP activity prediction, PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), is a bioactive peptide prediction server based on a novel N-to-1 neural network. Submitting a peptide list to PeptideRanker will rank the peptides according to their probability of biological activity, selecting 72 sequences with an activity probability ranging from 0 to 0.6. Higher activity indicates greater toxicity.

[0092] The secondary structure of 72 AMP sequences was predicted using the GOR4 online server (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_gor4.html). GOR4 is based on information theory and Bayesian statistics to predict the secondary structure of short sequences. - There are 48 sequences with a proportion of more than 60% alpha helix and random coil and less than 40% extended strand. If the percentage of extended strand in a sequence is high, it is not easy to maintain a stable spatial structure.

[0093] The isoelectric points of the remaining 48 sequences were predicted using PepCalc.com (https: / / pepcalc.com / ppc.php). Sequences with positive charges showed stronger membrane penetration capabilities. For example, the positive control peptide vector TAT-HA2 had an isoelectric point of 11.84 and carried 5 positive charges.

[0094] To predict sequence toxicity, the Fasta format of the AMP sequence is input into Toxinpred3.0 (https: / / webs.iiitd.edu.in / raghava / toxinpred3 / blast.php). Toxinpred3.0 allows users to search for the query protein sequence in a database of known toxic and non-toxic proteins using a similarity-based search method (i.e., BLAST). If the given query sequence receives any match from the database, this module predicts the query sequence as non-toxic; if it does not receive any match, it is predicted as non-toxic, and sequences with a toxicity rating of Non-Toxin are selected.

[0095] Finally, sequences containing fewer Q, N, C, D, and E amino acid residues are preferred.

[0096] As shown in Table 1, 23 AMP sequences were selected and linked to TAT (SEQ ID NO: 20-23 were not linked to TAT and served as controls), and the sequences were synthesized in reverse from the C-terminus to the N-terminus.

[0097] Table 1

[0098] Example 2: Experimental screening of AMP sequences with high delivery efficiency In this embodiment, the amino acid sequence of TAT in TAT-AMP is shown in SEQ ID NO: 25.

[0099] 1. TAT-AMP delivers Cas9 knockout EL4 mCherry and THP-1 mCherry Viral infection of EL4 and THP-1 cells: EL4 and THP-1 cells in logarithmic growth phase were seeded into 10 cm cell culture dishes. Cell counts were performed, and 200,000 cells were collected. Different lentiviral gradients (0.15-5 μL) were used to infect cells (lentiviruses containing U6-sgRNA-EFS-mCherry plasmid vector) using 12-well plates. 200,000 cells per well were added to the above-mentioned gradient doses of virus solution and 4 μL of polybrene (2 mg / mL). The medium was either EL4 mCherry complete medium (DMEM containing 10% FBS and 1% penicillin / streptomycin) or THP-1 mCherry complete medium (RPMI 1640 containing 10% FBS and 1% penicillin / streptomycin) to a final volume of 1 mL per well. After centrifugation at 558 × g for 25 min at room temperature, the cells were incubated statically at 37°C with 5% CO2. 24 hours after infection, the culture medium was changed to DMEM or RPMI 1640 complete medium and incubated at 37℃ with 5% CO2 to allow the plasmid to carry resistance expression. After 24 hours of static culture, Blastidin S-HCl was added to a final concentration of 10 μg / mL.

[0100] (1) The concentration of Cas9 protein was diluted to 10 μM using serum-free cell culture medium (DMEM medium for EL4 mCherry cells and RPMI 640 medium for THP-1 mCherry cells). (Cas9 protein was obtained by expression using the Cas9-T6N vector (https: / / www.addgene.org / 199604), which has TAT-4×Myc NLS at the N-terminus and 2×SV40 NLS-sfGFP at the C-terminus). (2) Dissolve the peptide in PBS solution to 1 mM, filter sterilize using a 0.22 μm filter, aliquot and store at -80℃. The peptide is a peptide composed of the C-terminus of TAT directly linked to the N-terminus of the AMP shown in SEQ ID NO: 1-19, and the amino acid sequence of TAT is as shown in SEQ ID NO: 24. Alternatively, the peptide is a single AMP shown in SEQ ID NO: 20-23. (3) Collect EL4 mCherry cells and THP-1 mCherry cells at 100-200K respectively, and centrifuge at 600×g for 3 min at room temperature to collect the cells. Wash the cells once with serum-free cell culture medium (DMEM medium and RPMI 640 medium respectively), and centrifuge at 600×g for 3 min at room temperature to collect the cells; (4) Add the diluted Cas9 to the tube, then add 20 μL of cell suspension, and finally add the peptide. Incubate at 37°C with 5% CO2 for 30 min (at this time, the concentration of Cas9 is 0.5 μM and the concentration of peptide is 50 μM). (5) Add 180 μL (10 times the volume) of complete culture medium to terminate the knockout (if cytotoxicity needs to be calculated, take 10 μL of cell suspension here for trypan blue staining and then cell counting), and collect cells by centrifugation at room temperature for 3 min at 600×g. (6) The cells were resuspended in 200 μL of complete culture medium and cultured in 96-well plates. The knockout efficiency was detected by flow cytometry on the sixth day.

[0101] The above-described process of TAT-AMP delivery of Cas9 knockout mCherry fluorescent protein in EL4 mCherry and THP-1 mCherry is as follows: Figure 1 As shown in the middle route.

[0102] Figure 2 The editing effects of TAT-HA2 (positive control peptide) and the peptide TAT-AMP (denoted as PeptideXX in the figure, for example, the peptide TAT-AMP composed of AMP and TAT linked together as shown in SEQ ID NO: 1 is denoted as Peptide1 in the figure, while Peptide20-23 refers only to the AMP shown in SEQ ID NO: 20-23) in EL4 mCherry cells were shown by flow cytometry. It can be seen that when using a final concentration of 50 μM peptide, the editing efficiency of TAT-HA2 is 63.5-76.4%, while Peptide4 and Peptide5 can achieve knockout efficiencies of 69.6-86.1% and 54.6-78.0%, respectively. The average efficiency of Peptide4 is about 9% higher than that of TAT-HA2.

[0103] Figure 3 The editing effects of TAT-HA2 (positive control peptide) and TAT-AMP (the peptide composed of AMP and TAT linked together in Table 1) on THP-1 mCherry cells were shown by flow cytometry. It can be seen that when using a final concentration of 50 μM peptide, Peptide 9 and Peptide 5 were slightly more effective than TAT-HA2, while Peptide 4, Peptide 10 and Peptide 18 all had an editing efficiency of about 80%, indicating that the TAT-AMP provided by this invention has a good editing effect on THP-1 mCherry cells.

[0104] 2. TAT-AMP delivery to RNP knockout Jurkat cells B2M Gene (1) Dilute the Cas12a protein concentration to 25 μM using serum-free cell culture medium (RPMI 640 medium); The Cas12a used in this application is a protein obtained by expression using the opCas12a-T8N (https: / / www.addgene.org / 199605 / ) vector, which has TAT-6×Myc NLS at the N-terminus and 2×SV40 NLS-sfGFP at the C-terminus; (2) Dilute crRNA to a concentration of 100 μM with DNase-free / RNase-free water, aliquot and store at -80℃; (3) Dissolve the peptides in PBS solution to 1 mM, filter sterilize using a 0.22 μm filter, aliquot and store at -80℃; (4) Dilute crRNA to a concentration of 20 μM using serum-free cell culture medium (RPMI 640 medium); (5) Dilute the peptide to a concentration of 50 μM using serum-free cell culture medium (RPMI 640 medium); (6) Mix the diluted Cas12a and diluted crRNA and let stand at room temperature for 20 min (to prepare RNP, at which time the concentration of crRNA is 10 μM and Cas12a is 12.5 μM, and the system volume is generally not less than 20 μL). (7) Mix 10 μL of RNP and 10 μL of peptide to make a total of 20 μL, and let stand at room temperature for 5 min (at this time, the concentration of peptide is 50 μM, crRNA is 5 μM, Cas12a is 6.25 μM, and RNP is 5 μM). (8) Take 100-200K cells and centrifuge at 600×g for 3 min at room temperature to collect the cells. Wash the cells once with serum-free cell culture medium (RPMI 640 medium) and centrifuge at 600×g for 3 min at room temperature to collect the cells; (9) Resuspend the cells in 20 μL of RNP+ polypeptide and incubate at 37℃ with 5% CO2 for 30 min. (10) Add 180 μL (10 times the volume) of complete culture medium (DMEM containing 10% FBS and 1% penicillin / streptomycin or RPMI 1640 containing 10% FBS and 1% penicillin / streptomycin) to terminate the knockout, and collect the cells by centrifugation at 600×g for 3 min at room temperature; (11) Resuspend the cells in 1 mL of complete culture medium, place them in a well plate for culture, and detect the knockout efficiency by flow cytometry on the sixth day.

[0105] The above-described process of delivering B2M to Cas12a knockout Jurkat cells is as follows: Figure 4 As shown in the middle route.

[0106] Figure 5 TAT-AMP delivery was demonstrated in RNP knockout Jurkat cells. B2M The process involves using engineered bacteria to express Cas12a protein with TAT and NLS fusion expression, which is then mixed with crRNA and TAT-AMP or AMP and incubated with cells to achieve efficient and relatively gentle entry into the cells, and finally efficient entry into the nucleus from the endosomes into the cytoplasm.

[0107] Figure 6 The above-mentioned technical solution was applied to the Jurkat cell line. B2M After gene knockout, flow cytometry analysis of surface B2M protein levels showed that using 50 μM TAT-HA2 resulted in an editing efficiency of 53.9-58.2%. Specifically, the editing efficiency for Peptide 4 reached 53.5-72.0%, and for Peptide 17, it reached 46.9-81.4%.

[0108] 3. TAT-AMP delivery to RNP knockout primary T cells B2M Gene Human primary T cells B2M The gene knockout method is the same as that used for Jurkat cells, except that the culture medium was changed to X-VIVO15 medium.

[0109] Figure 7 , 8 This refers to the editing effect of TAT-HA2 and TAT-AMP on human primary T cells. Figure 7 The results showed that at a final peptide concentration of 10 μM, TAT-HA2 achieved an editing efficiency of approximately 17%, while Peptide4 achieved an editing efficiency of 36.4-44.1%, with an average efficiency approximately 20% higher than TAT-HA2. At a final peptide concentration of 25 μM, TAT-HA2 achieved an editing efficiency of approximately 40%, while Peptide4 achieved an editing efficiency of approximately 50%, representing an improvement of approximately 10% compared to TAT-HA2.

[0110] Figure 9 This study tested the cytotoxicity of 25 μM final concentrations of TAT-HA2 and TAT-AMP on human primary T cells. After incubating cells with RNP and TAT-AMP for 30 min, 180 μL (10 times the volume) of complete culture medium was added to terminate the knockout. After mixing, 10 μL of cells was collected, stained with trypan blue, and the viable cell count was determined. Using the mock viable cell count as 100%, the average viability of TAT-HA2 cells was 72.2%, Peptide 4 cells were 70.5%, and Peptide 12 cells were 85.1%, indicating low cytotoxicity.

[0111] Figure 10The above method was applied to primary human T cells to... B2M On day 10 after gene knockout, flow cytometry was used to detect the surface B2M protein level. It was found that when using 25 μM TAT-HA2 and Peptide4, the editing efficiency was 36.6% and 43.9%, respectively.

[0112] 4. Detect the delivery efficiency of modified TAT-AMP or (Arg)9-AMP. Figure 11 The process involves replacing the N-terminus of TAT-AMP with (Arg)9 and then applying the above-mentioned "2. TAT-AMP delivery to RNP knockout Jurkat cells" to the Jurkat cell line. B2M In the "gene" technical scheme, after knocking out the B2M gene, the surface B2M protein level was detected by flow cytometry. It was found that without the use of peptides, the editing efficiency was only 0.69-2.68%. When using a final concentration of 25 μM peptides, the editing efficiency of Peptide38 (amino acid sequence as shown in SEQ ID NO: 38) reached 64.3-66.2%, and the editing efficiency of Peptide37 (amino acid sequence as shown in SEQ ID NO: 37) reached 25.8-27.8%, both significantly higher than the editing efficiency without peptides.

[0113] Figure 12 The editing effect in EL4 mChe267 cells was achieved by replacing the N-terminus of TAT-AMP with (Arg)9 and using the technique described in "1. TAT-AMP delivery of Cas9 knockout of EL4 mCherry and THP-1 mCherry". Without the peptide, the editing efficiency was only about 2.45%. However, with a final peptide concentration of 25 μM, the editing efficiency of Peptide37 reached approximately 49%, and that of Peptide38 reached approximately 20.5%, both significantly higher than the editing efficiency without the peptide.

[0114] Figure 13 The B2M gene was knocked out in Jurkat cell lines after acetylation modification of the C-terminus of TAT-AMP, and the surface B2M protein level was detected by flow cytometry. Using a final peptide concentration of 25 μM, the editing efficiency of Peptide34 (amino acid sequence shown in SEQ ID NO: 34) reached 31.3-34.9%, significantly higher than the editing efficiency of the group without peptide (only 0.69-2.68%).

[0115] Figure 14This describes the editing effect of acetylation of the C-terminus of TAT-AMP in EL4 mChe267 cells. Using a final peptide concentration of 25 μM, Peptide34 achieved an editing efficiency of approximately 30%, significantly higher than the editing efficiency without peptide (only 2.45%).

[0116] Figure 15 TAT-AMP was modified with N-terminal fatty acid and C-terminal acetylation, and the peptides were tested in the Jurkat cell line using the technique described in "1. TAT-AMP delivery of Cas9 knockout EL4 mCherry and THP-1 mCherry". At a final peptide concentration of 25 μM, the efficiency of Peptide35 (amino acid sequence as shown in SEQ ID NO: 35) was 5.65-6.37%, and that of Peptide36 (amino acid sequence as shown in SEQ ID NO: 36) was 10.8-11.7%, both showing delivery efficiency.

[0117] Figure 16 This is the editing result of TAT-AMP after N-terminal fatty acid modification and C-terminal acetylation modification in the EL4 mChe267 cell line. It is evident that Peptide35 has delivery efficiency.

[0118] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0119] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A polypeptide, characterized in that, It includes a cell-penetrating peptide and an antimicrobial peptide amino acid sequence, wherein the C-terminus of the cell-penetrating peptide is linked to the N-terminus of the antimicrobial peptide amino acid sequence; or, The C-terminus of the amino acid sequence of the antimicrobial peptide is linked to the N-terminus of the cell-penetrating peptide. The amino acid sequence of the antimicrobial peptide is as shown in SEQ ID NO: 1-19, or is a derived sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-19.

2. The polypeptide according to claim 1, characterized in that, The cell-penetrating peptide includes at least one selected from TAT, (Arg)9, Penetratin, GALA, and LAH4; Optionally, the cell-penetrating peptide is TAT ​​or (Arg)9.

3. The polypeptide according to claim 2, characterized in that, The amino acid sequence of the TAT is as shown in SEQ ID NO: 24 and SEQ ID NO: 25, or is a derived sequence having the same function as the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 25; The amino acid sequence of (Arg)9 is as shown in SEQ ID NO: 30, or is a derived sequence having the same function as the amino acid sequence shown in SEQ ID NO:

30.

4. The polypeptide according to any one of claims 1-3, characterized in that, At least one amino acid in the amino acid sequence of the cell-penetrating peptide and / or antimicrobial peptide is a D-type amino acid; Optionally, the amino acid sequences of the cell-penetrating peptide and / or antimicrobial peptide are unmodified or chemically modified; Optionally, the chemical modification includes amidation or sulfation of C-terminal amino acids; acetylation or fatty acidation of N-terminal amino acids; glycosylation of at least one of the intermediate residues serine, tyrosine, tryptophan, and asparagine; phosphorylation of at least one of the intermediate residues serine, tyrosine, and tryptophan; cyclization; and polyethylene glycol modification.

5. The polypeptide according to claim 2 or 3, characterized in that, In the polypeptide, the C-terminus of the amino acid sequence of TAT or (Arg)9 is directly linked to the N-terminus of the amino acid sequence of the antimicrobial peptide; or, The C-terminus of the antimicrobial peptide amino acid sequence is directly linked to the N-terminus of the amino acid sequence of TAT or (Arg)9. Optionally, the amino acid sequence of the TAT is as shown in SEQ ID NO: 24 or SEQ ID NO: 25; the amino acid sequence of the (Arg)9 is as shown in SEQ ID NO:

30. Optionally, the amino acid sequence of the antimicrobial peptide is as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO:

18. Optionally, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:

38.

6. Use of the polypeptide according to any one of claims 1-5 in improving the gene editing efficiency of the CRISPR / Cas system.

7. The use according to claim 6, characterized in that, The Cas protein involved in the CRISPR / Cas system is a CRISPR system effector protein; Optionally, the CRISPR system effector proteins include one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

8. Use of the polypeptide of any one of claims 1-5 in the preparation of reagents or kits for improving the gene editing efficiency of CRISPR / Cas systems.

9. The use according to claim 8, characterized in that, The Cas protein involved in the CRISPR / Cas system is a CRISPR system effector protein; Optionally, the CRISPR system effector proteins include one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

10. A reagent or kit for improving the gene editing efficiency of CRISPR / Cas systems, characterized in that, The polypeptide includes any one of claims 1-5.

11. A method for improving the gene editing efficiency of the CRISPR / Cas system, characterized in that, include: The polypeptide described in any one of claims 1-5 and the delivery cargo are mixed and introduced into the target cells. The delivered goods include: The target gene is either sgRNA or crRNA or a complex with a CRISPR system effector protein, or the CRISPR system effector protein itself.

12. The method according to claim 11, characterized in that, The CRISPR system effector proteins include one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

13. A fusion protein, characterized in that, The amino acid sequence of at least one polypeptide according to any one of claims 1-5.

14. The fusion protein according to claim 13, characterized in that, The C-terminus of the polypeptide is linked to the N-terminus of a CRISPR system effector protein; or, The N-terminus of the polypeptide is linked to the C-terminus of a CRISPR system effector protein. Optionally, the CRISPR system effector proteins include one or more selected from Cas12a, Cas9, Cas13, Cas3, Cas8, Csel, Csyl, Csn2, Cas4, Cas10, Csm2, Cmr5, and Fokl.

15. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein as described in claim 13 or 14.

16. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 15.

17. A recombinant virus, characterized in that, The recombinant virus comprises the expression vector of claim 16, and the recombinant virus is capable of infecting target cells.

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