A method for enhancing the nuclear localization ability of a crisper / cas system

Abstract

This invention relates to the field of biotechnology, specifically to a method for enhancing the nuclear localization capability of the CRISPR / Cas system. The invention provides a fusion protein and a method for enhancing the nuclear localization capability of the CRISPR / Cas system in target cells. The fusion protein provided by this invention comprises a cell-penetrating peptide (TAT), a Cas protein, and at least one amino acid sequence containing a nuclear localization signal. The inventors have discovered that a specific cell-penetrating peptide (TAT) and a specific nuclear localization signal, when combined with the Cas protein to form a fusion protein, enable highly efficient nuclear localization of the Cas protein with the assistance of the helper peptide TAT-HA2. This improves the accuracy and efficiency of gene editing, providing a safer and more efficient tool for future gene therapy and immunotherapy.

Description

[0001] Priority information This application claims priority and benefit to patent application 202510368113.5, filed with the China National Intellectual Property Administration on March 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a method for enhancing the nuclear localization capability of the CRISPR / Cas system. Background Technology

[0003] From its initial discovery to its functional elucidation and subsequent technological breakthroughs and innovations, the CRISPR / Cas system has demonstrated its crucial role in biology and gene editing. How to efficiently facilitate the nuclear translocation of Cas proteins to exert their target chain cleavage function is a key concern. The challenges to the safety and efficiency of CRISPR / Cas delivery systems primarily include ensuring the system is safely and effectively delivered to target cells and achieves its function without triggering an immune response. Existing technologies for delivering effector proteins from CRISPR / Cas systems into the cell nucleus for gene editing mainly include: lipid nanoparticles (LNPs), extracellular vesicles (EVs), polymer vesicles, protein / peptide delivery carriers, metal-organic frameworks (MOFs), mesoporous silica nanoparticles (MSNs), and physical methods. However, current delivery systems still have some drawbacks and technical difficulties, mainly in the following aspects: (1) Delivery efficiency and targeting Low delivery efficiency: Existing delivery technologies often struggle to ensure efficient and accurate protein delivery to target cells. This is likely due to the cell membrane's barrier effect on proteins, and the complex and difficult-to-control interaction between the delivery vehicle and the cell membrane. Insufficient targeting: During delivery, proteins may not be precisely located to target cells or tissues, but instead become widely distributed throughout the organism. This not only reduces the efficiency of gene editing but may also increase side effects and potential risks. (2) Cytotoxicity and immunogenicity Cytotoxicity: Some delivery vectors or methods may cause damage or toxicity to cells, affecting their normal function and survival. This limits the application of delivery technologies in the field of gene editing. Immunogenicity: The delivery carrier or protein itself may trigger an immune response in the body, leading to immune rejection or inflammation. This not only reduces delivery efficiency but may also harm the organism. (3) Stability and controllability Stability issues: During delivery, proteins may be affected by the biological environment, such as enzymatic degradation and oxidation, leading to reduced activity or inactivation. This limits the application of delivery technologies in long-term gene editing or complex biological environments. Poor controllability: Existing delivery technologies often struggle to precisely control the timing and amount of protein release. This can lead to unstable or unpredictable gene editing effects. (4) Technological complexity and cost Technical complexity: Some delivery technologies require delicate operations and sophisticated equipment support, which increases the difficulty and cost of implementing the technology.

[0004] High cost: Efficient delivery vectors or methods are often accompanied by high research and development and production costs, which limits their feasibility in clinical or widespread applications.

[0005] Therefore, there is an urgent need to develop a CRISPR / Cas delivery system with low cytotoxicity and high gene editing efficiency. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one object of this invention is to provide a fusion protein and a method for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells. The fusion protein provided by this invention includes a Cas protein and at least one amino acid sequence containing a nuclear localization signal. The inventors have discovered that a specific cell-penetrating peptide, TAT, combined with a specific nuclear localization signal, forms a fusion protein with the Cas protein. With the assistance of the helper peptide TAT-HA2, the Cas protein can achieve efficient nuclear localization, thereby improving the accuracy and efficiency of gene editing and providing a safer and more efficient tool for future gene therapy and immunotherapy.

[0007] Therefore, a first aspect of the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: The transmembrane peptide TAT, Cas protein, and at least one amino acid sequence containing a nuclear localization signal. The C-terminus of the Cas protein is linked to the N-terminus of the amino acid sequence containing the nuclear localization signal, and the N-terminus of the Cas protein is linked to the C-terminus of the transmembrane peptide TAT.

[0008] Existing CRISPR / Cas delivery systems still have some drawbacks and technical challenges. Optimizing the nuclear localization signal (NLS) in the CRISPR / Cas system to promote nuclear transport of Cas proteins and improve gene editing efficiency is a major challenge. The fusion protein provided in this invention solves the problem of nuclear localization delivery at the cellular level in CRISPR / Cas systems, ultimately improving primary cell gene editing efficiency, reducing cytotoxicity, and its safety has been assessed.

[0009] Specifically, the present invention is based on the following: Cell-penetrating peptides (CPPs) are a class of short peptides capable of penetrating cell membranes and delivering therapeutic carriers that are otherwise impermeable to the cell interior. CPPs can enter cells through various pathways, including direct transmembrane transport and endocytosis. CPPs facilitate the penetration of biomolecules across the cell membrane, increasing the likelihood of these macromolecules entering the cell. Nucleus-penetrating peptides (NLSs) typically consist of a string of positively charged amino acids (R and K), which allows them to be recognized and bound by transport proteins in the nucleus, thereby facilitating intranuclear protein transport. NLSs are crucial for ensuring that key proteins such as transcription factors and DNA repair enzymes accurately reach the nucleus to perform their biological functions.

[0010] Nuclear localization signals (NLS) are used in gene editing technology primarily because eukaryotic DNA is distributed within the cell nucleus, and gene editing often requires manipulation of this nuclear DNA. This invention, based on a peptide-based CRISPR / Cas delivery system, fuses an optimized NLS to enable the active transport of the Cas protein, a crucial component of the CRISPR / Cas system, to the cell nucleus in eukaryotic cells. CPP and NLS are two powerful biomolecular delivery systems that play key roles in crossing cell membranes and ensuring molecules reach specific cellular locations. CPP primarily involves cell membrane penetration and endocytosis, while NLS is specifically responsible for delivering proteins into the cell nucleus; the two are complementary and play crucial roles in intracellular molecule delivery and localization. The fusion protein provided by this invention contains an accessory peptide sequence, TAT, derived from the HIV virus-derived CPP sequence. This facilitates better cell membrane crossing and endosome escape.

[0011] According to an embodiment of the present invention, an amino acid sequence containing a nuclear localization signal is further linked between the transmembrane peptide TAT and the Cas protein, wherein the N-terminus of the Cas protein is linked to the C-terminus of the amino acid sequence containing the nuclear localization signal, and the N-terminus of the amino acid sequence containing the nuclear localization signal is linked to the C-terminus of the transmembrane peptide TAT.

[0012] According to an embodiment of the present invention, the amino acid sequence containing a nuclear localization signal attached to the C-terminus of the Cas protein and the amino acid sequence containing a nuclear localization signal attached to the N-terminus of the Cas protein may be the same or different.

[0013] According to an embodiment of the present invention, the Cas protein includes at least one selected from Cas12a, Cas9, Cas3, Cas14 and its derivatives.

[0014] According to an embodiment of the present invention, the derivative of Cas9 is the Cas9 adenine base editor ABE8e.

[0015] According to an embodiment of the present invention, the protein containing the nuclear localization signal sequence is derived from at least one of VP1, HTLV-1 Rex, Nucropialasmin, cMyc, SV40, ING4, p53, and BP.

[0016] According to embodiments of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 45.

[0017] A second aspect of the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect.

[0018] A third aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect.

[0019] A fourth aspect of the present invention provides a recombinant virus. According to an embodiment of the present invention, the recombinant virus comprises the expression vector described in the third aspect, and the recombinant virus is capable of infecting target cells.

[0020] A fifth aspect of the present invention provides a cell. According to an embodiment of the present invention, the cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or is capable of expressing the fusion protein described in the first aspect.

[0021] A sixth aspect of the present invention provides a composition. According to an embodiment of the present invention, the composition comprises one or more of the following: The fusion protein described in the first aspect; The nucleic acid molecules described in the second aspect; The expression carrier described in the third aspect.

[0022] The seventh aspect of the present invention provides the use of the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant virus described in the fourth aspect in enhancing the nuclear localization capability of the CRISPR / Cas system.

[0023] An eighth aspect of the present invention provides a composition or kit for enhancing the nuclear localization capability of the CRISPR / Cas system in target cells. According to an embodiment of the present invention, the composition or kit comprises: (1) Helper peptide TAT-HA2, and (2) The fusion protein described in the first aspect and / or the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect.

[0024] The gene editing tool delivery system provided by this invention includes the helper peptide TAT-HA2 and a Cas protein containing at least one nuclear localization signal sequence. The inventors have discovered that the specific cell-penetrating peptide TAT-HA2 and the specific nuclear localization signal enable the Cas protein to achieve efficient nuclear localization, thereby improving the accuracy and efficiency of gene editing and providing a safer and more efficient tool for future gene therapy and immunotherapy.

[0025] The ninth aspect of this invention provides the use of the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant virus described in the fourth aspect in the preparation of a kit that enhances the nuclear localization ability of the CRISPR / Cas system in target cells.

[0026] A tenth aspect of the present invention provides a method for enhancing the nuclear localization capability of the CRISPR / Cas system in target cells. According to an embodiment of the present invention, the method includes introducing the following into the target cells: a. The nucleic acid molecules described in the second aspect and / or the expression vector described in the third aspect and / or the infection of target cells using the recombinant virus described in the fourth aspect; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

[0027] The eleventh aspect of this invention provides a method for improving gene editing efficiency in target cells. According to an embodiment of the invention, the method includes introducing the following into the target cells: a. The nucleic acid molecules described in the second aspect and / or the expression vector described in the third aspect and / or the infection of target cells using the recombinant virus described in the fourth aspect; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

[0028] A twelfth aspect of the present invention provides a method for improving the knockout efficiency of a target gene in a target cell. According to an embodiment of the present invention, the method includes introducing the following into the cell: a. The nucleic acid molecules described in the second aspect and / or the expression vector described in the third aspect and / or the recombinant virus described in the fourth aspect to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c. crRNA, which is designed to target a gene.

[0029] The thirteenth aspect of this invention provides a method for improving the base editing efficiency of a target gene in a target cell. According to an embodiment of the invention, the method includes introducing the following into the cell: a. The nucleic acid molecules described in the second aspect and / or the expression vector described in the third aspect and / or the recombinant virus described in the fourth aspect to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c. sgRNA, wherein the sgRNA is designed to target a gene. 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

[0030] 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 Thirteen fusion protein structures according to embodiments of the present invention are shown; Figure 2a-2m The following are the protein electrophoresis images of the samples during the expression and purification of 6×cMyc-Cas12a-2×SV40, 6×cMyc-Cas12a-2×Rex, 6×cMyc-Cas12a-2×cMyc, 6×cMyc-Cas12a-2×NUC, 6×cMyc-Cas12a-2×VP1, 6×cMyc-Cas12a-2×ING4, 6×cMyc-Cas12a-2×p53, 6×cMyc-Cas12a-2×BP, 2×SV40-Cas12a-6×cMyc, 2×ING4-Cas12a-6×cMyc, 4×cMyc-Cas9-2×SV40, 4×cMyc-Cas9-2×ING4, and TAT-4×cMyc-TadA8e-nCas9-2×ING4 proteins. Figure 3aThe effects of eight protein forms—TAT-6×cMyc-Cas12a-2×p53, TAT-6×cMyc-Cas12a-2×VP1, TAT-6×cMyc-Cas12a-2×NUC, TAT-6×cMyc-Cas12a-2×cMyc, TAT-6×cMyc-Cas12a-2×Rex, TAT-6×cMyc-Cas12a-2×SV40, TAT-6×cMyc-Cas12a-2×ING4, and TAT-6×cMyc-Cas12a-2×BP—on target genes in Jurkat cells at different AP concentrations were shown. B2M Gene editing efficiency; Figure 3b The effects of three protein forms—TAT-6×cMyc-Cas12a-2×ING4, TAT-6×cMyc-Cas12a-2×BP, and TAT-6×cMyc-Cas12a-2×SV40—on Jurkat cell viability at different AP concentrations were shown. Figure 4a The effects of three protein forms—TAT-2×ING4-Cas12a-6×cMyc, TAT-2×SV40-Cas12a-6×cMyc, and TAT-6×cMyc-Cas12a-2×SV40—on the target gene at different AP concentrations were shown. B2M Gene editing efficiency; Figure 4b The effects of different AP concentrations on cell viability were shown for three protein forms: TAT-2×ING4-Cas12a-6×cMyc, TAT-2×SV40-Cas12a-6×cMyc, and the control TAT-6×cMyc-Cas12a-2×SV40. Figure 5a The effects of TAT-6×cMyc-Cas12a-2×ING4 protein on the target gene at different AP concentrations were shown. B2M Gene editing efficiency; Figure 5b The effect of TAT-6×cMyc-Cas12a-2×ING4 protein on cell viability at different AP concentrations was shown. Figure 6 The route for knocking out the target gene mCherry in EL4 or Jurkat-mCherry cells is shown; Figure 7aThe gene editing efficiency of the two protein forms, TAT-4×cMyc-Cas9-2×ING4 and TAT-4×cMyc-Cas9-2×SV40, on the target gene mCherry at different AP concentrations and in Jurkat-mCherry cells is shown in the top figure, as well as the gene editing efficiency on the target gene mCherry in Jurkat-Rosa cells (bottom figure). Figure 7b The effects of two protein forms, TAT-4×cMyc-Cas9-2×ING4 and TAT-4×cMyc-Cas9-2×SV40, on the survival rate of Jurkat cells at different AP concentrations were shown. Figure 8a The gene editing efficiency of the two protein forms, TAT-4×cMyc-Cas9-2×ING4 and TAT-4×cMyc-Cas9-2×SV40, on the target gene mCherry at different AP concentrations and in EL4-mCherry cells is shown in the top figure, as well as the gene editing efficiency on the target gene mCherry in EL4-Rosa cells (bottom figure). Figure 8b The effects of two protein forms, TAT-4×cMyc-Cas9-2×ING4 and TAT-4×cMyc-Cas9-2×SV40, on the survival rate of EL4 cells at different AP concentrations were shown. Figure 9a The effects of eight protein forms—TAT-6×cMyc-Cas12a-2×p53, TAT-6×cMyc-Cas12a-2×ING4, TAT-6×cMyc-Cas12a-2×BP, TAT-6×cMyc-Cas12a-2×cMyc, TAT-6×cMyc-Cas12a-2×Rex, TAT-6×cMyc-Cas12a-2×NUC, TAT-6×cMyc-Cas12a-2×VP1, and the control TAT-6×cMyc-Cas12a-2×SV40—on target genes in mouse primary CD8 T cells at different AP concentrations were shown. Thy1 Gene editing efficiency; Figure 9bThe effects of eight protein forms—TAT-6×cMyc-Cas12a-2×p53, TAT-6×cMyc-Cas12a-2×VP1, TAT-6×cMyc-Cas12a-2×Rex, TAT-6×cMyc-Cas12a-2×ING4, TAT-6×cMyc-Cas12a-2×cMyc, TAT-6×cMyc-Cas12a-2×BP, TAT-6×cMyc-Cas12a-2×NUC, and TAT-6×cMyc-Cas12a-2×SV40—on the survival rate of primary CD8 T cells in mice at different AP concentrations were shown. Figure 10a The effects of seven protein forms—TAT-6×cMyc-Cas12a-2×VP1, TAT-6×cMyc-Cas12a-2×NUC, TAT-6×cMyc-Cas12a-2×cMyc, TAT-6×cMyc-Cas12a-2×Rex, TAT-6×cMyc-Cas12a-2×BP, TAT-6×cMyc-Cas12a-2×ING4, and TAT-6×cMyc-Cas12a-2×SV40—on target genes at different AP concentrations were shown. B2M Gene editing efficiency; Figure 10b The effects of three protein forms—TAT-6×cMyc-Cas12a-2×ING4, TAT-2×ING4-Cas12a-2×cMyc, and TAT-6×cMyc-Cas12a-2×SV40—on the target gene at different AP concentrations were shown. B2M Gene editing efficiency; Figure 10c The effects of three protein forms—TAT-6×cMyc-Cas12a-2×ING4, TAT-2×ING4-Cas12a-2×cMyc, and TAT-6×cMyc-Cas12a-2×SV40—on cell viability at different AP concentrations were shown. Figure 11 The effects of TAT-4×cMyc-TadA8e-nCas9-2×ING4 protein on the target gene at different AP concentrations were shown. B2M Gene editing efficiency; Figure 12a The experimental procedure for peptide-mediated intramuscular delivery of Cas12a RNP is shown; Figure 12b This demonstrates the working principle of the Ai9 mouse TdTomato gene editing reporter system; Figure 12c The results of mouse muscle imaging are shown; Figure 12d Showing Figure 12c The results of quantitative analysis of fluorescence intensity. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0036] 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.

[0037] Currently, enhancing the nuclear localization of Cas proteins by adding nuclear localization signals (NLS) has become a common strategy. NLS, as specific sequences within nucleophiles, utilize their "localization" function to ensure the accurate transport of Cas proteins through the nuclear pore complex into the cell nucleus, thereby facilitating efficient nuclear entry into the CRISPR / Cas system for gene editing. While the addition of NLS can improve the nuclear localization of Cas proteins, the impact of the type, number, and location of NLS on gene editing efficiency has not been systematically studied. Different NLS sequences may possess different nuclear localization capabilities and effects on Cas protein activity, while the insertion location and number of NLS may also influence the structure and function of Cas proteins. This invention aims to optimize the type, number, and location of NLS to achieve efficient nuclear localization of Cas proteins in eukaryotic cells, thereby improving the accuracy and efficiency of gene editing and providing a safer and more efficient tool for future gene therapy and immunotherapy.

[0038] Compared with traditional delivery methods such as electroporation, viral vectors, and LNPs, peptide delivery CRISPR / Cas systems have a series of significant advantages, making them a powerful tool in gene editing technology. These advantages include: (1) non-viral delivery: compared with traditional viral vector delivery CRISPR / Cas systems, peptide delivery avoids the immune response and potential carcinogenic risks that may be caused by viral delivery, thus improving safety; (2) efficient transmembrane and nuclear penetration capabilities: peptide delivery CRISPR / Cas systems such as CPP and NLS can efficiently cross the cell membrane and enter the nucleus, achieving rapid and efficient intracellular delivery; (3) reduced cytotoxicity and side effects: compared with other delivery methods such as electroporation, peptide delivery has lower cytotoxicity and side effects, which is beneficial for maintaining the physiological state and function of cells. (4) Editing flexibility and diversity: Peptide delivery systems can be applied to different types of CRISPR / Cas systems (such as Cas9 and Cas12a), editing strategies (such as gene knockout, knock-in or base editing), and delivery forms (RNP or Cas protein), providing greater editing flexibility and diversity; (5) Convenient and quick preparation and modification: Peptide synthesis is more economical, simple and quick compared to the construction and production of viral vectors and LNPs, making it easy to quickly respond to different research needs and optimize delivery strategies.

[0039] Furthermore, effective nucleus spectroscopy (NLS) is one of the key factors for achieving efficient gene editing. NLS ensures that Cas proteins enter the cell nucleus, interact with target DNA, and perform cutting or modification functions, making it an indispensable step in the gene editing process. A lack of effective NLS or weak NLS signaling may limit the nuclear transport of Cas proteins, reducing the efficiency and specificity of gene editing. Therefore, NLS has a significant impact on optimizing the design of CRISPR / Cas systems and improving gene editing efficiency.

[0040] Based on this, the inventors conducted extensive experimental research and obtained the following special structure of the peptide delivery CRISPR / Cas system.

[0041] According to a specific embodiment of the present invention, the present invention provides a fusion protein, comprising: The transmembrane peptide TAT, Cas protein, and at least one amino acid sequence containing a nuclear localization signal. The C-terminus of the Cas protein is linked to the N-terminus of the protein containing the nuclear localization signal sequence, and the N-terminus of the Cas protein is linked to the C-terminus of the transmembrane peptide TAT.

[0042] According to a specific embodiment of the present invention, an amino acid sequence containing a nuclear localization signal is further linked between the transmembrane peptide TAT and the Cas protein, wherein the N-terminus of the Cas protein is linked to the C-terminus of the amino acid sequence containing the nuclear localization signal, and the N-terminus of the amino acid sequence containing the nuclear localization signal is linked to the C-terminus of the transmembrane peptide TAT.

[0043] According to a specific embodiment of the present invention, the amino acid sequence containing a nuclear localization signal attached to the C-terminus of the Cas protein and the amino acid sequence containing a nuclear localization signal attached to the N-terminus of the Cas protein may be the same or different.

[0044] It should be noted that the fusion protein includes at least one protein containing a nuclear localization signal sequence, and may also include two proteins containing nuclear localization signal sequences. When two proteins are included, the two proteins containing nuclear localization signal sequences may be the same or different.

[0045] According to a specific embodiment of the present invention, the protein containing the nuclear localization signal sequence is derived from at least one of VP1, HTLV-1 Rex, Nucropialasmin, cMyc, SV40, ING4, p53, and BP.

[0046] According to a specific embodiment of the present invention, the Cas protein includes at least one of the CRISPR-Cas family proteins selected from Cas12a, Cas9, Cas3, Cas14, etc., and their derivatives.

[0047] According to a specific embodiment of the present invention, the derivative of Cas9 is the Cas9 adenine base editor ABE8e.

[0048] According to a preferred embodiment of the present invention, the Cas protein is the Cas12a protein.

[0049] The reason is that the V-type CRISPR / Cas12a system has multiple advantages, such as smaller protein molecular weight than Cas9, higher editing specificity, and shorter corresponding crRNA (CRISPR RNA), which is less expensive and more suitable for artificial synthesis. These advantages are conducive to the clinical application of this system.

[0050] The fusion protein provided by this invention contains an accessory peptide AP sequence that includes the HIV-derived CPP sequence TAT, which helps the system to better cross cell membranes and escape endosomes. Specifically, TAT protein is a key regulatory protein in HIV-1, whose main function is to activate viral gene expression; TAT protein promotes viral gene transcription by binding to the transcriptional activation response element (TAR) of viral RNA; the TAT sequence can efficiently penetrate the cell membrane and enter the cell interior; the TAT peptide exhibits low immunogenicity and cytotoxicity during cell penetration.

[0051] According to a specific embodiment of the present invention, the fusion protein includes two proteins containing nuclear localization signal sequences.

[0052] According to a specific embodiment of the present invention, the fusion protein further comprises at least one cMyc protein.

[0053] According to a specific embodiment of the present invention, the C-terminus of the transmembrane peptide TAT is linked to the N-terminus of the Cas protein via at least one cMyc protein.

[0054] According to a specific embodiment of the present invention, the fusion protein further comprises six linked cMyc proteins.

[0055] According to a more specific embodiment of the present invention, the fusion protein may be an appendage. Figure 1 At least one of the following structures: (1)TAT-6×cMyc-Cas12a-2×ING4; (2) TAT-6×cMyc-Cas12a-2×BP; (3) TAT-2×ING4-Cas12a-6×cMyc; (4) TAT-4×cMyc-Cas9-2×ING4; (5)TAT-4×cMyc-ABE8e-nCas9-2×ING4.

[0056] It should be noted that in the structures (1)-(13) above, for example, “TAT-6×cMyc-Cas12a-2×ING4” means that the fusion protein is connected from the N-terminus to the C-terminus by TAT, 6 tandem cMyc, the amino acid sequence of Cas12a protein, and 2 tandem ING4. Other numbered fusion protein structures have the same meaning, the only difference being the difference in NLS name or Cas.

[0057] According to a specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 45.

[0058] According to a specific embodiment of the present invention, the fusion protein structure provided by the present invention is as follows: Figure 1 As shown.

[0059] According to a specific embodiment of the present invention, the present invention provides a nucleic acid molecule that encodes the aforementioned fusion protein.

[0060] According to a specific embodiment of the present invention, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0061] According to a specific embodiment of the present invention, the present invention provides a recombinant virus comprising the aforementioned expression vector, the recombinant virus being capable of infecting target cells. The recombinant virus may, for example, be an adenovirus, lentivirus, or retrovirus comprising the aforementioned expression vector.

[0062] According to a specific embodiment of the present invention, the present invention provides a cell that carries the aforementioned nucleic acid molecule or the aforementioned expression vector or is capable of expressing the aforementioned fusion protein. According to a specific embodiment of the present invention, the cell may be, for example, Jurkat cells, EL4 cells, and primary T cells or NK cells from humans and mice.

[0063] According to a specific embodiment of the present invention, the present invention provides a composition comprising one or more of the following: The fusion protein described above; The nucleic acid molecules mentioned above; The expression carriers mentioned above.

[0064] According to a specific embodiment of the present invention, the present invention provides a composition or kit for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells, comprising: (1) Helper peptide TAT-HA2, and (2) The fusion protein and / or the nucleic acid molecule and / or the expression vector described above.

[0065] In the composition or kit, the TAT protein promotes viral gene transcription by binding to the transcriptional activation response element (TAR) of viral RNA; the TAT sequence can efficiently penetrate the cell membrane and enter the cell interior; the TAT peptide exhibits low immunogenicity and cytotoxicity during cell penetration. The endosome escape peptide (EEP) sequence is derived from the HA2 fragment of influenza A virus hemagglutinin (HA). The HA2 sequence is pH sensitive and can induce membrane perturbations in acidic environments (such as endosomes or lysosomes), thereby helping carried molecules escape from endosomes and enter the cytoplasm. This property makes HA2 suitable for enhancing the endosome escape capability of drug or gene delivery systems. This invention provides a specific cell-penetrating peptide TAT and a special nuclear localization signal to form a fusion protein with the Cas protein. With the assistance of the helper peptide TAT-HA2, the Cas protein can achieve efficient nuclear localization, thereby improving the accuracy and efficiency of gene editing.

[0066] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells (such as Jurkat cells, primary T cells, NK cells, etc.), the method comprising introducing the following into the target cells: a. The fusion protein, nucleic acid molecule, and / or the expression vector described above, and / or the recombinant virus described in the fourth aspect, used to infect target cells; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

[0067] According to a specific embodiment of the present invention, the present invention provides a method for improving gene editing efficiency in target cells, the method comprising introducing into the target cells: a. The fusion protein, nucleic acid molecule, and / or the expression vector described above, and / or the recombinant virus described in the fourth aspect, used to infect target cells; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

[0068] According to a specific embodiment of the present invention, the present invention provides a method for improving the knockout efficiency of target genes in target cells, the method comprising introducing the following into the cells: a. The aforementioned fusion protein, nucleic acid molecule, and / or the aforementioned expression vector and / or the aforementioned recombinant virus used to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c. crRNA, which is designed to target a gene.

[0069] According to a specific embodiment of the present invention, the present invention provides a method for improving the base editing efficiency of a target gene in a target cell, the method comprising introducing into the cell: a. The aforementioned fusion protein, nucleic acid molecule, and / or the aforementioned expression vector and / or the aforementioned recombinant virus used to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c.sgRNA, wherein the sgRNA is designed to target the gene.

[0070] The following examples demonstrate how to construct Cas12a protein expression vectors with different NLS sequences, purify the corresponding fusion proteins, evaluate gene editing efficiency using flow cytometry, thereby determining the optimal Cas12a-NLS configuration, and finally verify gene editing efficiency and its safety in primary cells, including comprehensive testing for cytotoxicity, off-target effects, and immunogenicity.

[0071] According to a specific embodiment of the present invention, the present invention provides a method for in situ gene knockout in mouse muscle, the method comprising introducing the following into cells: a. The aforementioned fusion protein, nucleic acid molecule, and / or the aforementioned expression vector and / or the aforementioned recombinant virus used to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c. crRNA, which is designed to target a gene.

[0072] 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.

[0073] Example 1 Using In-Fusion cloning technology, according to Figure 1The design scheme was used to clone the Cas12a-CPP / NLS fusion protein expression vector and perform whole plasmid sequencing.

[0074] The plasmid vector used was Addgene #Plasmid #199605, https: / / www.addgene.org / 199605 / The inserted fragment is located between Cas12a and sfGFP. The inserted sequence is as follows: 2 × VP1 amino acid sequence: RRARRPRGEDRRARRPRG (SEQ ID NO: 1); The 2 × VP1 nucleic acid sequence is: AGACGAGCTCGTCGGCCCAGGGGAGAAGATCGCCGCGCGCGTCGTCCGCGTGGT (SEQ ID NO: 2) 2 × HTLV-1 Rex amino acid sequence: PKTRRRPRRSQRKRPPTEDPKTRRRPRRSQRKRPPT (SEQ ID NO: 3) The 2 × HTLV-1 Rex nucleic acid sequence is as follows: CCAAAAACACGTCGGCGACCCAGAAGGTCACAACGCAAGCGCCCACCGACCGAGGACCCGAAAACCCGTCGCCGTCCGCGTAGAAGCCAGCGTAAGCGTCCGCCGACG (SEQ ID NO: 4) 2 × Nuceloplasmin amino acid sequence: KRPAATKKAGQAKKKKEDKRPAATKKAGQAKKKK (SEQ ID NO: 5) The 2 × Nuceloplasmin nucleic acid sequence is as follows: AAGAGGCCCGCGGCAACAAAAAAAGCTGGACAAGCGAAAAAGAAGAAAGAGGACAAACGTCCGGCAGCTACCAAAAAGGCCGGTCAGGCGAAAAAGAAGAAG (SEQ ID NO: 6) The 2 × SV40 amino acid sequence is as follows: PKKKRKVEDPKKKRKV (SEQ ID NO: 7) The 2 × SV40 nucleic acid sequence is as follows: cctaagaaaaaacgaaaagttgaggatcctaaaaagaaacgaaaagtt (SEQ ID NO: 8) The 2 × cMyc amino acid sequence is as follows: PAAKRVKLDEDPAAKRVKLD (SEQ ID NO: 9) The 2 × cMyc nucleic acid sequence is as follows: cccgccgcgaagcgtgtaaagctggatgaggatccagcggcaaaaagggtgaaactagac (SEQ IDNO: 10) The amino acid sequence of 2 × ING4 is as follows: KGKKGRTQKEKKAARARSKGKNEDKGKKGRTQKEKKAARARSKGKN (SEQ ID NO: 11) The 2 × ING4 nucleic acid sequence is as follows: AAAGGTAAGAAAGGTCGGACACAGAAAGAGAAAAAGGCCGCCAGAGCAAGATCGAAGGGAAAAAATGAGGAAGAAGGGTCGCACCCAGAAGGAAAAGAAGGCTGCCCGGGCTCGCTCTAAAGGAAAAAAC (SEQ ID NO: 12) The amino acid sequence of 2 × p53 is as follows: KRALPNNTSSSPQPKKKPEDKRALPNNTSSSPQPKKKP (SEQ ID NO: 13) The 2 × p53 nucleic acid sequence is as follows: AAACGCGCCCTGCCTAACAATACCTCaTCCTCTCCTCAACCAAAgAAAAgCCTgaggacAAACGTGCTTCTTCCTAATAATAACTagcTCgTCTCCTCAACCTAAAAAgAAACCg (SEQ ID NO: 14) The 2×BP amino acid sequence is as follows: KRTADGSEFEPKKKRKVEDKRTADGSEFEPKKKRKV (SEQ ID NO: 15) The 2×BP nucleic acid sequence is: AAAAGGACAGCTGATGGATCAGAATTTGAGCCGAAAAAGAAGCGTAAGGTGGAGGATAAACGTACCGCGGATGGTAGCGAGTTCGAGCCGAAAAAGAAGCGCAAAGTT (SEQ ID NO: 16) The 4×cMyc amino acid sequence is as follows: PAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLD (SEQ ID NO: 17) The 4×cMyc nucleic acid sequence is as follows: ccggctgcaaagcgcgtcaaattggatggtgggtcacctgcggctaagcgagtcaaattagacggaggatcgcccgccgcgaagcgtgtaaagctggatgggggtagcccagcggcaaaaagggtgaaactagac (SEQ IDNO: 18) The 6×cMyc amino acid sequence is as follows: PAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLD (SEQ ID NO: 19) The 6×cMyc nucleic acid sequence is: ccggctgcaaagcgcgtcaaattggatggtgggtcacctgcggctaagcgagtcaaattagacggaggatcgcccgccgcgaagcgtgtaaagctggatgggggt agcccagcggcaaaaagggtgaaactagacggcggctccccggcagccaagagagtcaaactcgacgggggtagccctgctgctaaacgtgttaagcttgat (seq ID NO:20) Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×SV40 fusion protein (the amino acid sequence of sfGFP will be shown separately later) is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×SV40-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×Rex fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×Rex-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×cMyc fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×cMyc-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×NUC fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×NUC-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×VP1 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×VP1-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×ING4 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×ING4-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×p53 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×p53-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-6×cMyc-Cas12a-2×BP fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-6×cMyc-Cas12a-2×BP-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-2×SV40-Cas12a-6×cMyc fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-2×SV40-Cas12a-6×cMyc-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-2×ING4-Cas12a-6×cMyc fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-2×ING4-Cas12a-6×cMyc-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-4×cMyc-Cas9-2×SV40 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-4×cMyc-Cas9-2×SV40-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-4×cMyc-Cas9-2×ING4 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-4×cMyc-Cas9-2×ING4-sfGFP fusion protein is as follows: Express Figure 1 The amino acid sequence of the TAT-4×cMyc-TadA8e-nCas9-2×ING4 fusion protein is as follows: Express Figure 1 The nucleic acid sequence of the TAT-4×cMyc-TadA8e-nCas9-2×ING4-sfGFP fusion protein is as follows: The amino acid sequence of sfGFP is as follows: KGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO: 47) The correctly sequenced plasmid was transformed into *E. coli* Rosetta 2(DE3)pLysS, and clones were picked and amplified in TB (Terrific Broth) medium. When the bacterial OD600 was approximately 0.6, expression was induced with 0.5 mM IPTG at 18°C ​​for 12-16 hours. After induction, the bacteria underwent lysis, affinity chromatography, SUMO digestion, ion exchange chromatography, and gel filtration chromatography to obtain the Cas12a-CPP / NLS fusion protein with a purity >95%.

[0075] The methods for lysis, affinity chromatography, SUMO enzyme digestion, ion exchange chromatography, and gel filtration chromatography are as follows: 1. If using Ni-NTA agarose for affinity chromatography, the steps are as follows: (1) Thaw the cells in an ice-water bath for 20 minutes, and at the same time prepare lysis buffer (1× Tiechui lysis buffer, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM TCEP, 1× protease inhibitor, 1 mM MPMSF), adding 8 ml of lysis buffer per gram of cells; (2) Resuspend the bacterial cells in lysis buffer and lyse them on ice with stirring for 1 h; (3) Centrifuge at 16,000 g for 30 minutes at 4°C, collect the lysate supernatant, filter it with a 0.22 µM filter, and store the lysate on ice or in a 4°C cold room; (4) Prepare Ni-NTA agarose. The volume of Ni-NTA agarose required is 1 / 10 of the volume of the lysis supernatant. (5) Transfer the Ni-NTA agarose suspension to a 60 mL column and add 10 mL of 50% Ni-NTA agarose suspension to each column, which is 5 mL of agarose column volume; (6) Drain the column and immediately wash the Ni-NTA agarose with 15 ml of Cas washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl and 1 mM TCEP) for a total of 3 washes; (7) Add 50 ml of lysis buffer supernatant to each column, mix thoroughly with Ni-NTA agarose, and incubate at 4°C with slow rotation for 1 hour; (8) After incubation, drain the column and immediately wash the Ni-NTA agarose five times with 15 ml of Cas washing buffer. Then wash three times with 15 ml of 20 mM imidazole buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM TCEP and 20 mM Imidazole). Finally, elute five times (10 minutes each time) with 2 ml of 300 mM imidazole buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM TCEP and 300 mM Imidazole) and collect the eluent. (9) Add 4.5 mg of SUMO protease ULP1 and 0.15% NP-40 to the elution buffer, put it into a dialysis bag, and dialyze at 4°C for 12-18 hours in dialysis buffer (20 mM Tris-HCl, pH 8.0, 1 mM TCEP, 5% Glycerol, 500 mM NaCl).

[0076] If Strep-Tactin is used for affinity chromatography, the steps are as follows: (1)-(7) are the same as the steps in the cleavage and Ni-NTA agarose affinity chromatography, except that the Ni-NTA agarose used in affinity chromatography is replaced with Strep-Tactin packing material; (8) Wash 5 times immediately with 15 ml Cas washing buffer; (9) Add 5 ml of SUMO protease digestion buffer (30 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.15% NP40, 1 mM TCEP) and add 4.5 mg of SUMO protease ULP1. (10) Rotate at 4℃ for 12-18 hours. On the second day, wash the column with 5 ml of SUMO protease digestion buffer and collect the flow-through.

[0077] 2. Ion exchange chromatography (1) Prepare buffer C (20 mM Tris-HCl, pH 8.0, 1 mM TCEP, 5% Glycerol, 130 mM NaCl) by mixing 80 ml of buffer A (20 mM Tris-HCl, pH 8.0, 1 mM TCEP, 5% Glycerol, 2 M NaCl); (2) Prepare a HiTrap SP HP (5 ml) cation exchange column. First, wash the column with 25 ml of water, then wash twice with buffer C, 25 ml each time. Inject the protein sample after affinity chromatography into the HiTrap SP HP column using a syringe, and place the column on ice; (3) Connect the HiTrap SP HP (5 ml) cation exchange column to the ÄKTA™pure (cytiva, analysis software UNICORN™). Set the ÄKTA™ pure system pressure limit to 0.5 MPa and the column inlet pressure to 0.5 MPa; (4) Equilibrate the column with 25 ml of 80% buffer A and 20% buffer B at a rate of 2 ml / min; (5) Elute the protein with a gradient of buffer A+B, with the gradient of buffer B from 20% to 70%, at a rate of 2 ml / min, for a total elution volume of 50 ml, and collect 1 ml of the gradient-eluted protein sample. (6) Perform SDS-PAGE on the components under the UV peak to determine which components contain the target protein.

[0078] 3. Gel filtration chromatography (1) Collect the eluted samples containing the target protein after SDS-PAGE and centrifuge them at 4°C and 3850 g using ultrafiltration centrifuge tubes; (2) When the remaining protein solution is 250 µl after centrifugation, add 14.75 ml of buffer S200 (10 mM MEPES-Na, pH 7.0, 1 M NaCl, 5 mM MgCl2, 2 mM TCEP) to the ultrafiltration tube and continue centrifugation at 3850 g at 4°C. Repeat step (2) twice to ensure that the protein is completely in the buffer S200 environment. (3) Transfer the concentrated protein (about 0.25 ml) from step (2) to a new 1.5 ml tube, centrifuge at 16,000 g and 4°C for 10 minutes, and carefully and slowly transfer the supernatant to the new tube; (4) Connect the Superdex 200 Increase 10 / 300 GL column to the ÄKTA™ column; (5) Set the ÄKTA™ system pressure limit to 2 MPa and the column inlet pressure to 2 MPa; (6) Equilibrate the Superdex 200 Increase 10 / 300 GL column with 50 ml S200 buffer at a flow rate of 0.3 ml / min; (7) Wash the 0.5 ml sample loop with 5 ml of water and 5 ml of S200 successively; (8) Inject the sample from step (3) into a 0.5 ml sample loop. Elute the column with 50 ml of S200 buffer at a rate of 0.2 ml / min and collect 0.25 ml of the protein elution sample; (9) Perform SDS-PAGE on each component under UV light to determine which components contain the target protein.

[0079] (10) Collect the eluted samples with a target protein purity >90% after SDS-PAGE and centrifuge them at 4°C and 3850 g using ultrafiltration centrifuge tubes; (11) When the protein solution is reduced to 250 µl, add 14.75 ml of Cas protein preservation solution (580 mM KCl, 40 mM Tris pH 7.5, 20% Glycerol, 2 mM TCEP-HCl, 2 mM MgCl2) to the ultrafiltration tube and continue centrifugation at 3850 g at 4°C. Repeat step (11) twice to ensure that the protein is completely in the Cas protein preservation solution environment. (12) The concentrated Cas protein sample was aseptically filtered in a clean bench using a 0.22 µM filter, aseptically dispensed into 10 µL / tube, and stored in a -80℃ freezer for later use; the concentration of Cas protein was determined using the Bradford method.

[0080] Using the methods described above, the proteins 6×cMyc-Cas12a-2×SV40, 6×cMyc-Cas12a-2×Rex, 6×cMyc-Cas12a-2×cMyc, 6×cMyc-Cas12a-2×NUC, 6×cMyc-Cas12a-2×VP1, 6×cMyc-Cas12a-2×ING4, 6×cMyc-Cas12a-2×p53, 6×cMyc-Cas12a-2×BP, 2×SV40-Cas12a-6×cMyc, 2×ING4-Cas12a-6×cMyc, 4×cMyc-Cas9-2×SV40, 4×cMyc-Cas9-2×ING4, and TAT-4×cMyc-TadA8e-nCas9-2×ING4 were expressed and purified. The gel images of each sample during purification are shown below. Figure 2a-2m As shown, this indicates that the target protein was obtained.

[0081] Example 2: Detection of target gene knockout efficiency in Jurkat cell lines Detect the target gene according to the following steps ( B2M Gene knockout efficiency: (1) The concentration of the target protein obtained in Example 1 was diluted to 25 μM using serum-free cell culture medium (RPMI 1640); (2) Dilute the crRNA (crRNA sequence for the B2M gene: UAAUUUCUACUCUUGUAGAUAAUUCUCUCUCCAUUCUUCA, SEQ ID NO: 48, wherein the last three ribonucleotides at the 3' end of the sequence are modified with a 2'-O-methyl group on the sugar ring) with DNase-free / RNase-free water to a concentration of 100 μM, aliquot and store at -80°C; (3) Dissolve the helper peptide TAT-HA2 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 1640); (5) Dilute TAT-HA2 to concentrations of 50, 100 and 150 μM using serum-free cell culture medium (RPMI 1640); (6) Mix the diluted target protein 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 the concentration of target protein is 12.5 μM, and the volume is generally not less than 20 μL). (7) Mix 10 μL of RNP and 10 μL of TAT-HA2 to make a total of 20 μL, and let stand at room temperature for 5 min (at this time, the concentration of TAT-HA2 is 25, 50 and 75 μM, and the concentration of Cas12a RNP is 5 μM). (8) Take 100-200 K Jurkat 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 1640) and centrifuge at 600×g for 3 min at room temperature to collect the cells; (9) Resuspend the cells in 20 μL of RNP+TAT-HA2 mixture and incubate at 37℃ with 5% CO2 for 30 min; (10) Add 180 μL of complete culture medium (10% FBS, 1% Penicillin-Streptomycin, RPMI 1640) to terminate the reaction, and collect cells by centrifugation at 600×g for 3 min at room temperature; (11) Resuspend the cells in 1 ml of complete culture medium, culture them in a 24-well plate, and detect the knockout efficiency by flow cytometry after four days.

[0082] Figure 3a , Figure 3b The results show that, using the delivery method provided by this invention, in the Jurkat cell line, when the TAT-HA2 concentration is 50 μM, TAT-6×cMyc-Cas12a-2×ING4 achieves an editing efficiency of approximately 61% and a cell survival rate of approximately 55.5%; TAT-6×cMyc-Cas12a-2×BP achieves an editing efficiency of approximately 62% and a cell survival rate of approximately 66.5%; while the control TAT-6×cMyc-Cas12a-2×SV40 only achieves an editing efficiency of approximately 56.9%, with a reduced cell survival rate of approximately 62%. These data indicate that the editing efficiency of TAT-6×cMyc-Cas12a-2×ING4 is superior to that of the control TAT-6×cMyc-Cas12a-2×SV40, demonstrating that the NLS sequence combination provided by this invention has strong CRISPR nuclear localization capability.

[0083] Figure 4a , Figure 4bThe results showed that, using the delivery method provided by this invention, in the Jurkat cell line, when the TAT-HA2 concentration was 25 μM, TAT-2×ING4-Cas12a-6×cMyc achieved an editing efficiency of approximately 62.5% and a cell survival rate of approximately 80%; while the control TAT-6×cMyc-Cas12a-2×SV40 only achieved an editing efficiency of approximately 22% and a cell survival rate of approximately 79.3%; TAT-2×SV40-Cas12a-6×cMyc achieved an editing efficiency of approximately 2% and a cell survival rate of approximately 86.6%; when the TAT-HA2 concentration was 50 μM... At 75 μM, TAT-2×ING4-Cas12a-6×cMyc achieved an editing efficiency of approximately 92.7% and a cell survival rate of approximately 67%; while the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 76.7% and a cell survival rate of approximately 70%; the control TAT-2×SV40-Cas12a-6×cMyc achieved an editing efficiency of approximately 14.2% and a cell survival rate of approximately 84.7%. When the TAT-HA2 concentration reached 75 μM, TAT-2×ING4-Cas12a-6×cMyc achieved an editing efficiency of approximately 95.6% and a cell survival rate of approximately 57%; while the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 90.6% and a cell survival rate of approximately 54.7%. The efficiency of the gene editing tool delivery method provided by this invention is approximately 49.2%, compared to approximately 49.2% for the control group TAT-2×SV40-Cas12a-6×cMyc, with a cell viability of approximately 63.7%. This indicates that the gene editing tool delivery method provided by this invention has high gene editing efficiency and cell viability.

[0084] Example 3: Detection of target gene knockout efficiency in human primary NK cells The target gene in human primary NK cells was detected according to the following steps. B2M Gene knockout efficiency: (1) The concentration of the target protein obtained in Example 1 was diluted to 25 μM using serum-free cell culture medium (N500); (2) Dilute the crRNA (crRNA sequence for the B2M gene: UAAUUUCUACUCUUGUAGAUAAUUCUCUCUCCAUUCUUCA, SEQ ID NO: 48, wherein the last three ribonucleotides at the 3' end of the sequence are modified with a 2'-O-methyl group on the sugar ring) with DNase-free / RNase-free water to a concentration of 100 μM, aliquot and store at -80°C; (3) Dissolve the helper peptide TAT-HA2 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 (N500); (5) Dilute TAT-HA2 to concentrations of 50, 100 and 150 μM using serum-free cell culture medium (N500); (6) Mix the diluted target protein 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 the concentration of target protein is 12.5 μM, and the volume is generally not less than 20 μL). (7) Mix 10 μL of RNP and 10 μL of TAT-HA2 to make a total of 20 μL, and let stand at room temperature for 5 min (at this time, the concentration of TAT-HA2 is 25, 50 and 75 μM, and the concentration of Cas12a RNP is 5 μM). (8) Take 100-200 K human primary NK cells, centrifuge at 600×g for 3 min at room temperature to collect the cells. Wash the cells once with serum-free cell culture medium (N500), centrifuge at 600×g for 3 min at room temperature to collect the cells; (9) Resuspend the cells in 20 μL of RNP+TAT-HA2 mixture and incubate at 37℃ with 5% CO2 for 30 min; (10) Add 180 μL of 10% complete culture medium (9 mL of amplification medium (N500 NK cell serum-free medium + NK cell activator) + 1 mL of inactivated FBS) to terminate the reaction, and collect the cells by centrifugation at 600×g for 3 min at room temperature. (11) Resuspend the cells in 1 ml of 1% complete culture medium (2 ml of 5% complete culture medium + 8 ml of amplification culture medium), place them in a 24-well plate, and culture for four days. Flow cytometry is then used to detect the knockout efficiency. Figure 5a , 5b .

[0085] Figure 5a , Figure 5bThe results show that, using the delivery method provided by this invention, in primary human NK cells, when the TAT-HA2 concentration is 0 μM, 6×cMyc-Cas12a-2×ING4 achieves an editing efficiency of approximately 1.3% and a cell survival rate of approximately 76.5%; when the TAT-HA2 concentration reaches 25 μM, 6×cMyc-Cas12a-2×ING4 achieves an editing efficiency of approximately 16.6% and a cell survival rate of approximately 47.5%; when the TAT-HA2 concentration reaches 50 μM, 6×cMyc-Cas12a-2×ING4 achieves an editing efficiency of approximately 75.5% and a cell survival rate of approximately 31%; and when the TAT-HA2 concentration reaches 75 μM, 6×cMyc-Cas12a-2×ING4 achieves an editing efficiency of approximately 85.8% and a cell survival rate of approximately 14%. This indicates that this delivery method has broad application prospects in primary human NK cells.

[0086] Example 4: Detection of Cas9 gene knockout efficiency 1. The establishment of the mCherry reporting system, as follows Figure 6 As shown, a plasmid expressing the mCherry protein was transfected into EL4 or Jurkat cells using lentivirus, and the plasmid also expressed gRNA targeting mCherry. Cells were then incubated at 37°C with purified Cas9-CPP / NLS fusion protein and different concentrations of the accessory peptide AP. Incubate at C for 30 minutes. After incubation, wash the cells once with complete culture medium and continue culturing the cells in 96-well plates, passaged every 1-2 days. After 4-6 days, analyze the percentage of mCherry-negative cells using flow cytometry.

[0087] The knockout efficiency of Cas9 on the target gene mCherry was tested according to the following steps: (1) Dilute the target protein to 10 μM using serum-free cell culture medium (DMEM); (2) Dissolve AP in PBS solution to 1 mM, filter sterilize using a 0.22 μm filter, aliquot and store at -80℃; (3) Take 100-200K EL4 or Jurkat cell lines 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 for EL4 cells / RMPI 1640 for Jurkat cells) and centrifuge at 600×g for 3 min at room temperature to collect the cells; (4) Add the diluted protein to the tube, then add 20 μL of cell suspension, and finally add AP. Incubate at 37℃ with 5% CO2 for 30 min (at this time, the protein concentration is 0.5 μM, and the AP concentration is 25, 50 and 75 μM). (5) Add 180 μL (10 times the volume) of complete culture medium to terminate the knockout, and collect the cells by centrifugation at 600×g for 3 min at room temperature; (6) Resuspend the cells in 200 μL of complete culture medium, culture them in 96-well plates, and detect the knockout efficiency by flow cytometry after four days. Figures 7a-7b , Figures 8a-8b .

[0088] Figures 7a-7b The results showed that, using the delivery method provided by this invention, in Jurkat-mCherry cells, when the TAT-HA2 concentration was 0 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 3.18% and a cell survival rate of approximately 87.8%; when the TAT-HA2 concentration reached 25 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 28.1% and a cell survival rate of approximately 67%; when the TAT-HA2 concentration reached 50 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 59.2% and a cell survival rate of approximately 51.8%; when the TAT-HA2 concentration reached 75 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 69.1% and a cell survival rate of approximately 38.3%; when the TAT-HA2 concentration reached 0 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 69.1% and a cell survival rate of approximately 38.3%; when the TAT-HA2 concentration reached 0 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 3 ... At a TAT-HA2 concentration of 25 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 2.8% and a cell survival rate of approximately 80.75%. At a TAT-HA2 concentration of 25 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 27.1% and a cell survival rate of approximately 64%. At a TAT-HA2 concentration of 50 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 53.8% and a cell survival rate of approximately 49.5%. At a TAT-HA2 concentration of 75 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 61.8% and a cell survival rate of approximately 28.5%. This indicates that the gene editing tool delivery method provided by this invention has high gene editing efficiency. Furthermore, no knockout efficiency was detected in the negative control Jurkat-Rosa26 cells. Figure 7a (See the lower half of the image) to demonstrate the specificity of mCherry knockout.

[0089] Figure 8a , Figure 8b The results showed that, using the delivery method provided by this invention, in EL4-mCherry cells, when the TAT-HA2 concentration was 0 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 4.3% and a cell survival rate of approximately 90%; when the TAT-HA2 concentration reached 25 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 48.2% and a cell survival rate of approximately 71%; when the TAT-HA2 concentration reached 50 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 60.2% and a cell survival rate of approximately 49%; and when the TAT-HA2 concentration reached 75 μM, TAT-4×cMyc-Cas9-2×ING4 achieved an editing efficiency of approximately 70% and a cell survival rate of approximately 30%, which is superior to the control TAT-4×cMyc-Cas9-2×SV40. This indicates that the gene editing tool delivery method provided by this invention has high gene editing efficiency. When the TAT-HA2 concentration was 0 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 3.6% and a cell survival rate of approximately 86.4%; when the TAT-HA2 concentration reached 25 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 36.6% and a cell survival rate of approximately 70%; when the TAT-HA2 concentration reached 50 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 51.1% and a cell survival rate of approximately 44%; when the TAT-HA2 concentration reached 75 μM, the control TAT-4×cMyc-Cas9-2×SV40 achieved an editing efficiency of approximately 62% and a cell survival rate of approximately 30%. This indicates that the gene editing tool delivery method provided by this invention is also applicable to Cas9 and has high gene editing efficiency. Furthermore, no knockout efficiency was detected in the negative control EL4-Rosa26 cells. Figure 8a (See the lower half of the image) to demonstrate the specificity of mCherry knockout.

[0090] Example 5: Detection of target gene knockout efficiency in primary mouse T cells Detect the target gene according to the following steps ( Thy1 Gene knockout efficiency: (1) The concentration of the target protein obtained in Example 1 was diluted to 10 μM using serum-free cell culture medium (RPMI 1640); (2) Dilute crRNA with DNase-free / RNase-free water (for targeting) Thy1The crRNA sequence of the gene is: UAAUUUCUACUCUUGUAGAUGUUCACCAGGCAGGCUGUCA, SEQ ID NO: 49, wherein the last three ribonucleotides at the 3' end of the sequence are modified with a 2'-O-methyl group on the sugar ring to a concentration of 100 μM, and stored at -80℃ after aliquoting. (3) Dissolve the helper peptide TAT-HA2 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 1640); (5) Dilute TAT-HA2 to concentrations of 10, 20 and 50 μM using serum-free cell culture medium (RPMI 1640); (6) Mix the diluted target protein and the diluted crRNA and let stand at room temperature for 20 min; (7) Mix 10 μL of RNP and 10 μL of TAT-HA2 to a total of 20 μL, and let stand at room temperature for 5 min; (8) Collect 100-200 K mouse primary T cells, 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 1640), centrifuge at 600×g for 3 min at room temperature to collect the cells; (9) Resuspend the cells in 20 μL of RNP+TAT-HA2 mixture and incubate at 37℃ with 5% CO2 for 30 min; (10) Add 180 μL of complete culture medium (10% FBS, 1% Penicillin-Streptomycin, RPMI 1640, 200 mM L-glutamine, 100× non-essential amino acids, 55 mM 2-mercaptoethanol, 1 M HEPES, 100 mM Sodium Pyruvate) to terminate the reaction, 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, culture them in a 24-well plate, and detect the knockout efficiency by flow cytometry after four days. Figures 9a-9b .

[0091] Figure 9a , Figure 9bThe results showed that, using the delivery method provided by this invention, in primary mouse T cells, when the TAT-HA2 concentration was 0 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 24.5% and a cell survival rate of approximately 84%; when the TAT-HA2 concentration reached 25 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 74.2% and a cell survival rate of approximately 65%; when the TAT-HA2 concentration was 0 μM, the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 17.7% and a cell survival rate of approximately 85%; when the TAT-HA2 concentration reached 25 μM, the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 69.2% and a cell survival rate of approximately 58%. This indicates that the gene editing tool delivery method provided by this invention has high gene editing efficiency and is also applicable to primary mouse T cells.

[0092] Example 6: Detection of target gene knockout efficiency in human primary T cells The target gene in human primary T cells was detected according to the following steps. B2M Gene knockout efficiency: (1) The concentration of the target protein obtained in Example 1 was diluted to 25 μM using serum-free cell culture medium (RPMI 1640); (2) Dilute the crRNA (crRNA sequence for the B2M gene: UAAUUUCUACUCUUGUAGAUAAUUCUCUCUCCAUUCUUCA, SEQ ID NO: 48, wherein the last three ribonucleotides at the 3' end of the sequence are modified with a 2'-O-methyl group on the sugar ring) with DNase-free / RNase-free water to a concentration of 100 μM, aliquot and store at -80°C; (3) Dissolve the helper peptide TAT-HA2 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 1640); (5) Dilute TAT-HA2 to concentrations of 50, 100 and 150 μM using serum-free cell culture medium (RPMI 1640); (6) Mix the diluted target protein 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 the concentration of target protein is 12.5 μM, and the volume is generally not less than 20 μL). (7) Mix 10 μL of RNP and 10 μL of TAT-HA2 to make a total of 20 μL, and let stand at room temperature for 5 min (at this time, the concentration of TAT-HA2 is 25, 50 and 75 μM, and the concentration of Cas12a RNP is 5 μM). (8) Take 100-200 K human primary T cells, 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 1640), centrifuge at 600×g for 3 min at room temperature to collect the cells; (9) Resuspend the cells in 20 μL of RNP+TAT-HA2 mixture and incubate at 37℃ with 5% CO2 for 30 min; (10) Add 180 μL of complete culture medium (5% FBS, X-VIVO medium, 10 μM NAC, 50 μM BME) to terminate the reaction, and collect cells by centrifugation at 600×g for 3 min at room temperature; (11) Resuspend the cells in 1 ml of complete culture medium (5% FBS, X-VIVO medium, 10 μM NAC, 50 μM BME, 100 U / mL recombinant human IL-2), and culture them in 24-well plates. After six days, flow cytometry was used to detect the knockout efficiency. Figures 10a-10c .

[0093] Figure 10a The results showed that, using the delivery method provided by this invention, in human primary T cells, when the TAT-HA2 concentration reached 0 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 10.5%; TAT-2×ING4-Cas12a-6×cMyc achieved an editing efficiency of approximately 8%, and the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 9.9%; when the TAT-HA2 concentration reached 25 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 72.9%, TAT-2×ING4-Cas12a-6×cMyc achieved an editing efficiency of approximately 74%, and the control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 61.7%; when the TAT-HA2 concentration reached 50 μM... At μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 89%, and TAT-2×ING4-Cas12a-6×cMyc also achieved an editing efficiency of approximately 89%, compared to approximately 84.9% for TAT-6×cMyc-Cas12a-2×SV40.

[0094] Figure 10b , Figure 10cThe results showed that when the TAT-HA2 concentration reached 0 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 6.5% and a cell survival rate of approximately 90%. TAT-2×ING4-Cas12a-6×cMYc achieved an editing efficiency of approximately 8.0% and a cell survival rate of approximately 90.5%. The control TAT-6×cMyc-Cas12a-2×SV40 achieved an editing efficiency of approximately 8.6% and a cell survival rate of approximately 70%. When the TAT-HA2 concentration reached 25 μM, TAT-6×cMyc-Cas12a-2×ING4 achieved an editing efficiency of approximately 70.7% and a cell survival rate of approximately 76.5%. TAT-2×ING4-Cas12a-6×cMYc achieved an editing efficiency of approximately 74% and a cell survival rate of approximately 80%. Compared with TAT-6×cMyc-Cas12a-2×SV40, the editing efficiency reached about 66%, and the cell survival rate was about 48.5%; indicating that the gene editing tool delivery method provided by the present invention has a high gene knockout efficiency in primary human T cells.

[0095] Example 7: Detection of the base editing efficiency of Cas9-ABE8e on the target gene Detect the target gene according to the following steps ( B2M Gene knockout efficiency: (1) The concentration of the target protein obtained in Example 1 was diluted to 25 μM using serum-free cell culture medium (RPMI 1640); (2) Dilute sgRNA (sgRNA sequence targeting the B2M gene) with DNase-free / RNase-free water. 5'-ACUCACGCUGGAUAGCCUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3', SEQ ID NO: 50, wherein the first three ribonucleic acid units at the 5' end of this sequence are modified with 2'-O-methyl groups on the sugar ring and the phosphate esters in the backbone are modified to thiophosphate esters, and the last three ribonucleic acid units at the 3' end of this sequence are also modified as described above) to a concentration of 100 μM, aliquoted and stored at -80°C; (3) Dissolve the helper peptide TAT-HA2 in PBS solution to 1 mM, filter sterilize using a 0.22 μm filter, aliquot and store at -80℃; (4) Dilute sgRNA to a concentration of 20 μM using serum-free cell culture medium (RPMI 1640); (5) Dilute TAT-HA2 to a concentration of 100 μM using serum-free cell culture medium (RPMI 1640); (6) Mix the diluted target protein and diluted sgRNA and let stand at room temperature for 20 min (to prepare RNP, at which time the concentration of crRNA is 10 μM and the concentration of target protein is 12.5 μM, and the volume is generally not less than 20 μL). (7) Take 100-200 K human primary T cells, 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 1640), centrifuge at 600×g for 3 min at room temperature to collect the cells; (8) Resuspend the cells in 10 μL of RNP, then add 10 μL of AP, and incubate at 37°C with 5% CO2 for 30 min. (At this time, the concentration of AP is 50 μM, sgRNA is 5 μM, and the target protein is 6.25 μM.) (8) Add 180 μL of complete culture medium (5% FBS, X-VIVO medium, 10 μM NAC, 50 μM BME) to terminate the knockout, and collect the cells by centrifugation at 600 × g for 3 min at room temperature.

[0096] (9) Resuspend the cells in 1 mL of complete culture medium (5% FBS, X-VIVO medium, 10 μM NAC, 50 μM BME, 100 U / mL recombinant human IL-2), and culture them in well plates. Repeat this method every two days to perform the cell knockout experiment again, for a total of three deliveries. Six days later, flow cytometry was used to detect the knockout efficiency. Figure 11 .

[0097] Figure 11 The results showed that the knockout efficiency was most significantly improved when the second delivery was achieved in primary human T cells using the delivery method provided by this invention, approximately doubling. When the TAT-HA2 concentration reached 50 μM, the editing efficiency of TAT-4×cMyc-TadA8e-nCas9-2×ING4 reached approximately 10.8%, indicating that the gene editing tool delivery method provided by this invention can also deliver the Cas9-ABE8e adenine base editor.

[0098] Example 8: Detection of the knockout efficiency of Cas12a RNP delivered by peptide vector on TdTomato in Ai9 mouse muscle. (1) The Cas12a protein expressed and purified by Escherichia coli was diluted to 40 μM with RPMI 1640 medium; (2) Dilute the crRNA (crRNA sequence targeting the stop cassette gene: UAAUUUCUACUCUUGUAGAUUCCAAACUCAUCAAUGUAUC, SEQ ID NO: 51, wherein the last three ribonucleotides at the 3' end of the sequence are modified with a 2'-O-methyl group on the sugar ring) to 100 μM with DNase-free / RNase-free water, aliquot and store at -80 °C. (3) Dissolve the polypeptide carrier in PBS to 1 mM, sterilize by 0.22 μm filter membrane, aliquot and store at -80 °C; (4) Dilute 100 μM crRNA to 40 μM with RPMI 1640; (5) Dilute the 1 mM polypeptide carrier to 300 μM using the same method; (6) Mix 40 μM Cas12a and 40 μM crRNA at a volume ratio of 1:1 and let stand at room temperature for 20 min to form RNP; (7) Then mix 20 μM RNP with 300 μM peptide carrier at a volume ratio of 1:1 and let stand at room temperature for 5 min; (8) The Ai9 mouse was gently held with its head down. After disinfecting the skin of the left lower abdomen with 75% ethanol, 0.3% sodium pentobarbital (50 mg / kg) was injected intraperitoneally. - ¹) Administer anesthesia; (9) Once the anesthesia takes effect, disinfect the skin on the lower leg and inject 50 μL of RNP-peptide complex intramuscularly; return to the cage for routine feeding after the operation. (10) On the seventh day, the mice were euthanized, and the calf muscles were placed in a small animal in vivo optical imaging system (PerkinElmerIVIS® Spectrum system) to detect TdTomato fluorescence signals, such as... Figure 12b , 12c .

[0099] Figure 12a The experimental procedure for peptide-mediated intramuscular delivery of Cas12a RNP was shown: Cas12a RNP was mixed with peptide and injected into the muscle tissue of Ai9 mice. The fluorescence intensity of TdTomato was then detected by small animal in vivo imaging. Figure 12b The working principle of the Ai9 mouse TdTomato gene editing reporter system is shown: In Ai9 mice, there is a stop cassette before the TdTomato gene, flanked by loxP sites, which inhibits gene expression under normal conditions; after the stop cassette is cleaved by Cas12a RNP, the stop signal is released, and the TdTomato gene can be expressed. Figure 12cThe results of mouse muscle imaging were shown: at a TAT-HA2 concentration of 150 μM, the TdTomato fluorescence intensity of the calf muscles of mice in the TAT-2×ING4-Cas12a-6×cMyc injection group was better than that of the control group TAT-6×cMyc-Cas12a-2×SV40. Figure 12d Yes Figure 12c Quantitative analysis of fluorescence intensity showed that the fluorescence editing efficiency of the TAT-2×ING4-Cas12a-6×cMyc treatment group was approximately 1.4 times that of the control group TAT-6×cMyc-Cas12a-2×SV40. These results indicate that the gene editing tool delivery method provided by this invention can also be achieved in vivo.

[0100] 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.

[0101] 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 fusion protein, characterized in that, include: The transmembrane peptide TAT, Cas protein, and at least one amino acid sequence containing a nuclear localization signal. The C-terminus of the Cas protein is linked to the N-terminus of the amino acid sequence containing the nuclear localization signal, and the N-terminus of the Cas protein is linked to the C-terminus of the transmembrane peptide TAT.

2. The fusion protein according to claim 1, characterized in that, The transmembrane peptide TAT and the Cas protein are further linked by an amino acid sequence containing a nuclear localization signal, wherein the N-terminus of the Cas protein is linked to the C-terminus of the amino acid sequence containing the nuclear localization signal, and the N-terminus of the amino acid sequence containing the nuclear localization signal is linked to the C-terminus of the transmembrane peptide TAT. Optionally, the amino acid sequence containing the nuclear localization signal attached to the C-terminus of the Cas protein and the amino acid sequence containing the nuclear localization signal attached to the N-terminus of the Cas protein may be the same or different.

3. The fusion protein according to claim 1, characterized in that, The Cas protein includes at least one selected from Cas12a, Cas9, Cas3, Cas14 and its derivatives; Optionally, a derivative of Cas9 is the Cas9 adenine base editor ABE8e.

4. The fusion protein according to claim 1, characterized in that, The amino acid sequence containing the nuclear localization signal is derived from at least one of VP1, HTLV-1 Rex, Nucroplasmin, cMyc, SV40, ING4, p53, and BP.

5. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO:

45.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1-5.

7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant virus, characterized in that, The recombinant virus comprises the expression vector of claim 7, and the recombinant virus is capable of infecting target cells.

9. A cell, characterized in that, The cell carries the nucleic acid molecule of claim 6 or the expression vector of claim 7 or the fusion protein of any one of claims 1-5.

10. A composition, characterized in that, The composition contains one or more of the following: The fusion protein according to any one of claims 1-5; The nucleic acid molecule according to claim 6; The expression vector according to claim 7.

11. Use of the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, and the recombinant virus of claim 8 in enhancing the nuclear localization capability of the CRISPR / Cas system.

12. A composition or kit for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells, characterized in that, include: (1) Helper peptide TAT-HA2, and (2) The fusion protein of any one of claims 1-5 and / or the nucleic acid molecule of claim 6 and / or the expression vector of claim 7.

13. Use of the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, and the recombinant virus of claim 8 in the preparation of a kit for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells.

14. A method for enhancing the nuclear localization ability of the CRISPR / Cas system in target cells, characterized in that, The method includes introducing the following into the target cells: a. The nucleic acid molecule of claim 6 and / or the expression vector of claim 7 and / or the recombinant virus of claim 8 used to infect target cells; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

15. A method for improving gene editing efficiency in target cells, characterized in that, The method includes introducing the following into the target cells: a. The nucleic acid molecule of claim 6 and / or the expression vector of claim 7 and / or the recombinant virus of claim 8 used to infect target cells; and b. The helper peptide TAT-HA2 and / or an expression vector capable of expressing the helper peptide TAT-HA2.

16. A method for improving the knockout efficiency of target genes in target cells, characterized in that, The method includes introducing the following into the cells: a. The nucleic acid molecule of claim 6 and / or the expression vector of claim 7 and / or the recombinant virus of claim 8 used to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c. crRNA, which is designed to target a gene.

17. A method for improving the base editing efficiency of target genes in target cells, characterized in that, The method includes introducing the following into the cells: a. The nucleic acid molecule of claim 6 and / or the expression vector of claim 7 and / or the recombinant virus of claim 8 used to infect target cells; b. The accessory peptide TAT-HA2 and / or an expression vector capable of expressing the accessory peptide TAT-HA2; and c.sgRNA, wherein the sgRNA is designed to target the gene.

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