Method for performing gene editing on cells

Abstract

Provided is a composition for performing gene editing on cells. The composition comprises: a nickase complex or an encoding nucleic acid molecule thereof, wherein the nickase complex comprises a nickase, an optional guide nucleic acid molecule, and a first recruitment component; a target nucleic acid molecule containing a target site homologous sequence, wherein the target nucleic acid molecule can be linked to or bind to a second recruitment component, the first recruitment component and the second recruitment component are suitable for forming specific binding, the nickase complex is suitable for generating a nick in a single strand of DNA in a genome without cleaving the double strands of the DNA, and the nickase complex is configured to be linked to the first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, wherein the target nucleic acid molecule is configured to be linked to or bind to the second recruitment component, and the first recruitment component and the second recruitment component are suitable for forming specific binding.

Description

Methods for gene editing of cells Technical Field

[0001] This invention relates to the field of biotechnology, specifically to methods for gene editing of cells and methods for constructing recombinant immune cells and their applications, and more specifically to compositions for gene editing of cells, gene-edited cells, methods for gene therapy of patients, methods for constructing transgenic organisms, methods for constructing recombinant immune cells, cells obtained by such methods, and their applications. Background Technology

[0002] Gene editing is a technology that alters an organism's genetic information by directly modifying its DNA sequence. In recent years, systems such as CRISPR-Cas9 have become star technologies in the field of gene editing due to their high efficiency and simplicity. CRISPR-Cas9 utilizes guide RNA (gRNA) to precisely locate the target gene sequence and then uses the Cas9 enzyme to cut it, triggering DNA repair mechanisms to insert, delete, or replace genes. This technology has broad application prospects in medicine, agriculture, and biological research, such as treating hereditary diseases and cancer, and improving crop varieties. Gene editing can correct or compensate for diseases caused by abnormal genes, offering significant hope for the treatment of genetic diseases and cancer. However, traditional gene editing methods often face challenges, such as the high frequency of random integration, which can lead to unintended consequences such as gene damage, insertional mutations, or oncogene activation. These risks limit the safety and effectiveness of gene editing in gene therapy and cell therapy.

[0003] Currently, these limitations are typically overcome by precisely controlling the insertion site of therapeutic genes in the genome through programmable integration, thereby minimizing the risks associated with random integration. However, current programmable gene editing technologies, typically represented by CRISPR / Cas9, cut both strands of DNA, creating double-strand breaks (DSBs) that trigger homology-directed repair (HDR) and other DNA repair pathways. These DSB-based gene editing methods can lead to serious adverse consequences, such as high-frequency base deletions or insertions, translocations, p53 pathway activation, and increased tumorigenesis risk. Furthermore, studies have shown that the application of non-viral programmable double-strand break-based gene knock-in technologies in immune cells (such as T cells) can reduce cell viability and induce the activation of stress pathways such as cGAS-STING, becoming a significant problem to be addressed in this field. These adverse consequences also pose significant safety risks in treatment.

[0004] To avoid the risks associated with DNA double-strand breaks, some methods use Cas9 nickase (nCas9), which cuts only one strand of DNA, for gene editing. However, these methods have low insertion efficiency and are difficult to effectively achieve various gene editing tasks, such as inserting large gene fragments. Other methods combine CRISPR tools with integrase, but these methods are complex to design and usually involve cuts on both strands of DNA, also posing a risk of DSB (Double Straining of DNA).

[0005] Cell therapy involves endowing autologous or allogeneic cells with new capabilities for treating a variety of diseases. This includes stem cell therapy, where stem cells are used for tissue repair and regeneration due to their potential for self-renewal and multi-lineage differentiation. Cell therapy can also include immunocellular therapy, such as chimeric antigen receptor T-cell (CAR-T) therapy, which uses genetically engineered, allogeneic or patient-associated T cells to enhance their ability to recognize and attack cancer cells. It can also include armored cell therapy, which modifies immune cells to utilize endogenous gene regulatory mechanisms to express genes that enhance anti-tumor responses, such as immunomodulatory factors, at the tumor site.

[0006] Non-viral, programmable large-fragment insertion can improve cell-based therapies such as CAR-T and TCR-T cell engineering. It can enhance the stability and function of engineered immune cells while reducing the risk of random insertion. However, current large-fragment insertion methods primarily rely on DNA double-strand cutting techniques, which increase the risks of genomic instability, toxicity, and translocation. The gene-editing method proposed in this application avoids double-strand cutting, providing a safer option for T-cell engineering. However, current cell therapies suffer from long manufacturing cycles and high costs.

[0007] Therefore, there is an urgent need to develop new gene editing techniques and new cell therapy methods to improve the effectiveness of cell therapy. Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0009] In a first aspect of this application, a composition for gene editing of cells is provided. According to embodiments of this application, the composition comprises: a nicking enzyme complex or its encoding nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule containing a target site homologous sequence, the target nucleic acid molecule being connectable to or bound to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding, wherein the nicking enzyme complex is adapted to generate a single-stranded nick in the genome, without simultaneously cleaving a double-stranded DNA, the nicking enzyme complex being configured to be connectable to the first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be connectable to or bound to the second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

[0010] According to embodiments of this application, by employing this composition, gene editing can be effectively achieved at specific locations by utilizing the single-strand break (SSB) repair mechanism of the gene editing system, based on the nicking enzyme activity of single-strand cutting, and by improving the homologous recombination / homologous repair efficiency using the target nucleic acid molecule as a template. This effectively avoids the adverse consequences caused by DNA double-strand cutting, and also effectively avoids the problem that single-strand breaks (SSBs) can be quickly repaired back to an unedited state, making it difficult to apply to gene editing alone.

[0011] In a second aspect of this application, a method for gene editing of cells is provided. According to an embodiment of this application, the method includes: contacting the composition described in the first aspect of this application with cells to introduce into the cells an enzyme encoding a nick enzyme complex and a target nucleic acid molecule for gene editing at least one given site of the cells.

[0012] According to the embodiments of this application, by employing this method, based on the nicking enzyme activity of single-strand cutting, the single-strand break (SSB) repair mechanism of cells can be utilized. The target nucleic acid molecule contains homologous sequences, which can be used as templates for homologous recombination / homologous repair at specific locations. This can effectively achieve gene editing at specific locations and effectively avoid the adverse consequences caused by DNA double-strand cutting. At the same time, it effectively avoids the problem that it is difficult to apply to gene editing alone due to the rapid repair of single-strand breaks (SSBs).

[0013] In a third aspect of this application, a method for long-fragment gene editing of cells is provided. According to embodiments of this application, the method includes: contacting the composition described in the first aspect of this application with cells to introduce a nick enzyme complex and a target nucleic acid molecule into the cells.

[0014] The target nucleic acid molecule includes an insert fragment and homologous sequences located on both sides of the insert fragment. The length of the insert fragment is not less than 100 bp, for example, not less than 200 bp, not less than 300 bp, not less than 500 bp, not less than 1 kb, and can even be as high as at least 10 kb.

[0015] In a fourth aspect of this application, a method for gene therapy on a patient is proposed. According to embodiments of this application, the method includes: determining the sequences of a target nucleic acid molecule and a guide nucleic acid molecule based on the patient's target gene; contacting the composition described in the first aspect of this application with cells from the patient, or introducing an enzyme encoding a nick enzyme complex and a target nucleic acid molecule into the cells via the methods described in the second and third aspects of this application, to perform gene editing at at least one given site in the cells. The method according to embodiments of this application can effectively perform targeted repair on the patient's cells, thereby improving the efficiency of disease treatment.

[0016] In a fifth aspect of this application, the use of the composition described in the first aspect of this application or gene-edited cells obtained by the methods of the second and third aspects of this application in the preparation of a medicament for the treatment and / or prevention of disease. According to embodiments of this application, targeted repair of a patient's cells can be effectively achieved, thereby improving the efficiency of disease treatment.

[0017] In a sixth aspect of this application, a method for constructing a gene-edited organism is provided. According to embodiments of this application, the method includes: contacting a composition described in the first aspect of this application with a cell, or introducing into the cell an enzyme encoding a nick enzyme complex and a target nucleic acid molecule using the methods described in the second and third aspects of this application, to perform gene editing at at least one given site in the cell, and obtaining the gene-edited organism based on the edited cell.

[0018] In a seventh aspect of this application, a gene-edited cell is disclosed. According to embodiments of this application, the cell is obtained by the methods described in the second and third aspects of this application, or by gene editing of cells using the composition described in the first aspect of this application.

[0019] In an eighth aspect of this application, a gene-edited cell population is provided. According to embodiments of this application, the cell population is obtained by the methods described in the second and third aspects of this application, or by gene editing of the cell population using the composition described in the first aspect of this application.

[0020] In a ninth aspect of this application, a genetically modified organism (GMO) is proposed. According to an embodiment of this application, the GMO is constructed using the method of the sixth aspect.

[0021] According to embodiments of this application, the inventors have discovered a novel molecular mechanism that alters the repair preference of a single DNA gap (SSB), thereby proposing a novel gene editing system and developing a highly efficient and low-risk programmable gene editing technology based on a single DNA single-strand cut. Traditional programmable gene editing, represented by CRISPR / Cas9 technology, creates DNA double-strand breaks (DSBs) by cutting both strands of DNA, thereby stimulating homology-directed repair (HDR) and other DNA repair pathways to achieve gene editing. These DSB-based gene editing methods often lead to adverse consequences, such as high-frequency base deletions or insertions, translocations, p53 pathway activation, and an increased risk of tumorigenesis. These adverse consequences still pose significant safety risks in treatment, greatly limiting the industrial application of gene editing, especially in meeting the needs of gene therapy. To avoid the risks associated with DNA double-strand breaks, some methods use Cas9 with no nuclease activity (dead Cas9, dCas9) or Cas9 nickase (nCas9) that only cuts one strand of DNA for gene editing. However, these methods have low efficiency for inserting large fragments, making it difficult to effectively insert large gene fragments and hindering practical industrial applications. Other methods combine CRISPR tools with integrase, but these methods are complex to design and often involve cuts on both strands of DNA, posing a risk of DSB (double-strand break) and are also unsuitable for industrial applications. Furthermore, studies have shown that non-viral programmable DSB-based gene knock-in technologies used on immune cells (such as T cells) can reduce cell viability and activate stress pathways such as cGAS-STING, posing certain safety risks and further complicating industrial applications. Through extensive experiments and experimentation with various technical approaches, the inventors of this application have discovered a novel molecular mechanism that promotes single-strand break-mediated gene insertion dependent on donor homologous sequences. Based on this mechanism, they have developed a novel, highly efficient, and low-risk gene editing method suitable for large fragment insertion. This method can achieve excellent gene insertion precision while avoiding the risks caused by DNA double-strand breaks, thereby enabling them to promote the application of this method in the treatment of genetic diseases and cancer.For example, in the treatment of genetic diseases, this gene-editing system has the ability to achieve durable treatment of genetic diseases by integrating the normal version of a defective gene into a specific location in the genome, inserting the coding DNA of a normal transcript (such as complementary DNA, cDNA) into a "safe harbor" site (integration into a genomic region that does not disrupt the basic gene function), or directly inserting it into an endogenous site of the defective gene. In the field of cancer treatment, this method can precisely insert immune-regulating genes into safe target sites to improve the safety and efficacy of advanced cancer treatments such as CAR-T or TCR-T cell therapy. Furthermore, the inventors of this application unexpectedly discovered that recombinant cells obtained using the above method exhibit higher survival rates, cell viability, and proliferation rates, improving the efficiency of in vivo or in vitro gene editing of cells, thereby enhancing the overall efficiency and safety of cell therapy. It also helps to increase the yield and quality of recombinant cells, making them more suitable for industrial production.

[0022] According to another aspect of this application, a novel method for constructing recombinant immune cells is proposed. This method does not induce DSB (dysplasia of the stem cell), effectively avoiding the difficulties and pain points of existing technologies, and providing a low-mutation-risk alternative for T-cell engineering. It is based on the following discoveries of the inventors:

[0023] Immunotherapy such as CAR-T has become an important strategy for cancer treatment. However, the random integration of CAR genes mediated by traditional lentiviral or retroviral vectors can lead to adverse reactions such as insertion mutations, heterogeneous expression levels, and potential oncogenicity. Recent studies have reported cases of secondary T-cell lymphoma following CAR-T therapy, some of which are directly related to CAR-carrying T cells. To address these issues, non-viral programmable CAR gene integration at pre-defined genomic sites offers a safer alternative.

[0024] Several cases of T-cell carcinogenesis following CAR-T therapy have been reported. The exact mechanism of secondary T-cell carcinogenesis remains unclear, but one potential cause is insertional mutations in T cells. In most reported cases, the vector integration site is located in genes with unclear oncogenicity, making it difficult to confirm the causal relationship between the vector and tumor development. A case of gastrointestinal T-cell lymphoma developed several weeks after BCMA CAR-T therapy (ciltacabtagene autoleucel, cilta-cel) was reported; the development of this tumor may be related to the integration of the CAR vector into the classic tumor suppressor gene TP53 (encoding the p53 protein). Furthermore, genes such as SSU72 may also drive this process.

[0025] Currently, non-viral programmable gene knock-in technology is believed to optimize immunocellular therapies such as CAR-T and TCR-T by improving the efficiency of engineered immune cells and reducing the risk of random integration. However, current site-specific gene knock-in methods mainly rely on cell engineering techniques based on DNA double-strand breaks (DSBs), which increases genomic instability, cytotoxicity, and translocation risk. Existing studies have shown that the application of non-viral programmable DSB-based gene knock-in technology in immune cells (such as T cells) reduces cell viability and induces activation of stress pathways such as cGAS-STING, becoming a significant problem to be solved in this field. Furthermore, the preparation of universal immunotherapies, such as universal CAR-T, requires gene editing at multiple sites; however, existing DSB-based gene editing methods increase the proportion of chromosomal translocations, large chromosomal deletions, and genomic rearrangements.

[0026] Furthermore, during in-depth research, the inventors discovered that transfection using dsDNA donors in DSB-based gene editing technology significantly leads to T cell death, affecting cell viability and proliferation rate, primarily due to the activation of the cGAS-STING pathway and numerous double-strand breaks in the cytoplasm. Recent studies have proposed improving T cell survival rates by co-transfecting dsDNA donors with Cas9 / gRNA ribonucleoprotein (RNP) complexes or by optimizing electroporation conditions; however, low cell survival rates when using dsDNA donors remain a major challenge in T cell engineering. Specifically, the direct transfer of DNA donors and the activation of the cGAS-STING pathway caused by the numerous double-strand breaks generated are among the main reasons for the low efficiency of non-viral programmable CAR-T cell preparation. To this end, the inventors of this application systematically optimized the delivery system to reduce activation of this pathway. By co-transfecting the DNA donor with a nick enzyme complex, they significantly reduced double-strand breaks, enhanced the efficiency of DNA donor delivery into the nucleus, and reduced cytoplasmic aggregation, thereby lowering cGAS-STING activation levels, reducing cell death rates, and ultimately improving the survival, activity, and proliferation rate of immune cells such as T cells. This method also reduces the risks of genomic instability, cytotoxicity, and translocations caused by DSB-based gene editing methods. It avoids chromosomal translocations and deletions due to double-strand breaks during multi-gene editing, significantly improving the engineering safety of immune cells, especially universal immune cells (such as universal CAR-T cells) required for multi-site editing. This innovative method develops a non-viral, programmable gene knock-in technology independent of double-strand breaks, which is of great significance for the preparation of immune cells such as CAR-T cells. It not only helps to improve cell yield and quality but also further enhances the overall efficiency and safety of cell therapy.

[0027] Therefore, this application proposes a method for constructing recombinant immune cells in vivo or in vitro. According to an embodiment of this application, the method includes: treating cells with a gene editing composition to perform targeted gene editing on at least one given site of the cells to obtain edited recombinant immune cells. The gene editing composition includes: a nicking enzyme complex or its encoding nucleic acid molecule, the nicking enzyme complex including a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule, the target nucleic acid molecule being connectable to or bound to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding, wherein the nicking enzyme complex is adapted to generate a single-stranded nick in the genome, without simultaneously cleaving a double-stranded DNA, the nicking enzyme complex being configured to be connectable to the first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be connectable to or bound to the second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

[0028] According to embodiments of this application, the recombinant immune cells obtained by the above method can effectively avoid the adverse consequences caused by DNA double-strand cutting, such as additional chromosomal translocations and inversions caused by DNA double-strand cutting. It also effectively promotes homologous recombination repair based on single-strand breaks, solving the problem of low gene insertion efficiency caused by the rapid repair of single-strand breaks (SSBs) back to an unedited state, making them difficult to apply alone to gene editing. According to embodiments of this application, the resulting recombinant immune cells (such as CAR-T cells) can effectively target and kill cancer cells, exhibiting comparable or better killing activity than CAR-T cells produced by lentiviral methods. It also avoids a series of problems caused by random integration of lentiviruses, such as insertional mutations, chromosomal instability, and heterogeneous expression caused by random insertion, reducing the risk of secondary T-cell carcinogenesis and improving safety. Furthermore, the inventors of this application unexpectedly discovered that the recombinant immune cells obtained by the above method exhibit higher survival rates, cell viability, and proliferation rates. This is of great significance for the engineering of immune cells, especially the preparation of CAR-T cells, as it can improve cell yield and quality, thereby enhancing the efficiency and safety of the entire cell therapy.

[0029] In another aspect of this application, a recombinant immune cell is provided, which is obtained by the method described in the first aspect.

[0030] In another aspect of this application, a population of recombinant immune cells is provided, said recombinant immune cells being obtained by the method described in the first aspect.

[0031] In other aspects of this application, the use of immune cells or immune populations obtained by the above methods in the preparation of pharmaceuticals for treating diseases, including genetic abnormalities such as gene mutation-related diseases, cancer, and autoimmune diseases.

[0032] It should be noted that the features and advantages described in this article are mutually applicable, and will not be repeated unless otherwise specified.

[0033] 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

[0034] 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:

[0035] Figure 1 illustrates the principles of DSB-based and SSB-based gene editing according to embodiments of this application. The left side of Figure 1 shows two common repair pathways after DNA double-strand breaks. DNA double-strand breaks are severe injuries in cells, primarily repaired through emergency non-homologous end joining (NHEJ), which usually results in base insertion or deletion mutations at the break point. In a few specific cases (such as during the G2 and S phases of the cell), the broken DNA can be precisely repaired via homologous recombination (HR) using homologous chromosomes as templates. The right side of Figure 1 demonstrates that after a DNA single-strand break, because the other strand remains intact, it can be used as a template for rapid and precise repair of the break.

[0036] Figure 2 shows a schematic diagram illustrating how recruiting target nucleic acid molecules containing homologous sequences of the target site to nick sites can enhance homologous recombination / homologous repair efficiency according to an embodiment of this application.

[0037] Figure 2 shows the target nucleic acid molecule (donor DNA) with homologous sequences at both ends (homologous arms, indicated in black, the same below) and the foreign nucleic acid sequence (in gray, indicated below) that is to be inserted into the specified site. This invention enables the nicking enzyme to specifically recruit the target nucleic acid molecule (recruitment components are indicated by crescent shapes and small gray dots, the same below). By increasing the concentration of target nucleic acid molecules containing homologous sequences to the target site near the nick site, the bias of SSB repair is altered, improving the efficiency of homologous recombination / homologous repair at the nick site (dark gray cells in the right figure represent cells that have precisely inserted foreign nucleic acid through homologous recombination / repair; gray cells represent wild-type cells with the same genotype at the specified site as before editing, the same below).

[0038] Figure 3 illustrates the novel molecular mechanism by which the inventors of this application discovered altering the SSB repair pathway preference. The left figure illustrates the repair pathway of SSB single-strand nick repair under normal circumstances. After an SSB single-strand nick occurs, because the other strand remains intact, the DNA strand containing the single-strand nick (SSB) is rapidly repaired using the other strand as a template, resulting in a precisely repaired original sequence without causing additional mutations. This application finds that increasing the concentration of target nucleic acid molecules containing homologous sequences to the target site near the nick site can significantly alter the SSB repair pathway preference, improve the efficiency of homologous recombination / homologous repair at the nick site, and achieve efficient and low-risk gene editing.

[0039] Figure 4 compares the differences between DSB-based gene editing using nucleases and the enIE (enhanced nickase-initiated editing) gene editing repair pathway proposed in this application, which uses SSB-based nickases.

[0040] The left panel of Figure 4 shows that after a nuclease that cuts the DNA double strand causes a double-strand break at the target site, it mainly repairs the break through imprecisely via NHEJ, easily producing non-target products with base insertion or deletion mutations. The right panel of Figure 4 shows that the enIE gene editing method of this application uses a nicking enzyme to cause a single-strand break in DNA, which increases the concentration of target nucleic acid molecules containing homologous sequences of the target site near the nick site, changes the preference of SSB repair, and improves the efficiency of homologous recombination / homologous repair at the nick site. In addition, sites that did not insert the target nucleic acid sequence through homologous recombination / homologous repair can be repaired back to the original sequence using the undamaged strand as a template and can be edited again.

[0041] Figure 5 shows a schematic diagram of recruiting multiple target nucleic acid molecules containing homologous sequences of the target site to the nick site to enhance editing efficiency through an amplification mechanism according to an embodiment of this application.

[0042] The upper part of Figure 5 shows the pathway of further recruiting target nucleic acids by guide nucleic acid molecules to increase the concentration of target nucleic acid molecules containing homologous sequences of the target site near the nick site, thereby further improving the efficiency of homologous recombination / homologous repair; the lower part of Figure 5 shows the pathway of further recruiting target nucleic acids by nicking enzymes to increase the concentration of target nucleic acid molecules containing homologous sequences of the target site near the nick site, thereby further improving the efficiency of homologous recombination / homologous repair.

[0043] Figure 6 shows a schematic diagram of a scheme for multiple editing (taking secondary editing as an example) according to an embodiment of this application.

[0044] In Figure 6 (left), after the initial editing using nucleases that cleave DNA double strands, cells where foreign nucleic acids were not accurately inserted typically exhibited mutations at the designated sites (shown as gray raised areas, the same below), disrupting the recognition sequence of guide nucleic acid molecules or proteins. This makes it difficult to improve the efficiency of accurate insertion during subsequent editing. In contrast, Figure 6 (right) shows that, according to the method of this application, using nick enzymes for editing, cells where the exogenous nucleic acid was not accurately inserted after the first editing remain wild-type and can be edited again, improving the overall efficiency of precise editing through homologous recombination / homologous repair.

[0045] Figure 7 shows a schematic diagram and editing effect of gene editing using the gene editing system of this application according to one embodiment of this application.

[0046] Figure 8 illustrates the effect of using the gene editing system of this application to improve the safety of gene editing according to one embodiment of this application.

[0047] Figure 9 shows the effect of repeating gene editing using the gene editing system of this application according to one embodiment of this application.

[0048] Figure 10 shows the effect of gene editing using the gene editing system of this application to amplify the recruitment effect according to one embodiment of this application.

[0049] Figure 11 illustrates the application of the gene editing system of this application in the treatment of genetic diseases according to one embodiment of this application: a gene fragment that can be used for treatment is integrated into a safe site, and normal gene expression is restored in a cell model simulating the disease.

[0050] Figure 12 illustrates the application of the gene editing system CRISPR enIE according to one embodiment of this application in the field of immune cell engineering: the gene editing system of this application has a higher cell viability when treating primary T cells; and it increases the number of cells with large DNA fragment insertions in human primary T cells.

[0051] The left figure in Figure 13 shows the ratio of gene editing efficiency of the gene editing system of this application to that of Cas9 when performing gene editing at two gene sites simultaneously, according to an embodiment of this application. The right figure shows that Cas9 caused chromosomal translocations when performing gene editing at two sites simultaneously, while the gene editing system of this application did not cause any detectable chromosomal translocations.

[0052] Figure 14 shows the efficiency ratio of inserting therapeutic genes into safe sites using the gene editing system of this application versus the Cas9 system according to Example 14 of this application.

[0053] Figure 15 shows the gene expression level after a therapeutic gene is inserted into a safe site using the gene editing system of this application according to one embodiment of this application.

[0054] Figure 16 shows the efficiency ratio of inserting therapeutic gene (LIPA cDNA) into a safe site in a cell line carrying a pathogenic mutation gene using the gene editing system of this application versus the Cas9 system, according to one embodiment of this application.

[0055] Figure 17 shows the expression level of a normal gene with LIPA cDNA inserted into its natural site using the gene editing system of this application according to one embodiment of this application.

[0056] Figure 18 shows the comparison results of cell viability of recombinant immune cells obtained according to Example 24 of this application.

[0057] Figure 19 shows the comparison results of the proliferation curves of recombinant immune cells obtained according to Example 25 of this application.

[0058] Figure 20 shows a comparison of high-throughput sequencing and RT-qPCR results obtained according to Example 26 of this application.

[0059] Figure 21 shows a comparison of the editing efficiency of the gene editing systems CRISPR enIE and Cas9 obtained according to Example 27 of this application.

[0060] Figure 22 shows the results of the efficiency improvement of the optimized CRISPR enIE and CRISPR enIE2 obtained according to Embodiment 28 of this application.

[0061] Figure 23 shows the results of the cytotoxicity test of CAR-T cells prepared by CRISPR enIE according to Example 29 of this application. Detailed Implementation

[0062] 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.

[0063] 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.

[0064] Composition for gene editing of cells

[0065] In a first aspect of this application, a composition for gene editing of cells is provided. According to embodiments of this application, the composition comprises: a nicking enzyme complex or its encoding nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule containing a target site homologous sequence, the target nucleic acid molecule being connectable to or bound to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding, wherein the nicking enzyme complex is adapted to create a single-stranded nick in the genome, without simultaneously cleaving double-stranded DNA, the nicking enzyme complex being configured to be connectable to the first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be connectable to or bound to the second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

[0066] According to embodiments of this application, the cell is a cell carrying a pathogenic gene in vivo. According to embodiments of this application, the gene editing is used to repair the pathogenic gene. According to embodiments of this application, the pathogenic gene is a single-gene disease or a polygenic disease pathogenic gene. According to embodiments of this application, the gene editing is performed in vitro within cells or in vivo. Thus, according to embodiments of this application, genetically related diseases can be treated or reversed through site-specific gene editing. Specifically, many genetic diseases are associated with multiple pathogenic variations in specific genes; for example, more than 250 IL2RG gene mutations have been reported in X-linked severe combined immunodeficiency (X-SCID). A promising universal treatment strategy is to integrate transgenes capable of complementing disease mutations into safe harbor or native sites, thereby providing stable and sustained gene expression for therapeutic applications. LAL-D is an autosomal recessive genetic disorder caused by mutations in the LIPA gene, which encodes lysosomal acid lipase. LAL-D leads to lipid accumulation in various tissues, resulting in a range of symptoms from liver dysfunction to cardiovascular risk. The most common mutation leading to LAL-D occurs at the splicing site of exon 8 of the LIPA gene (LIPA894G>A), which causes the exon 8 to be excluded during RNA splicing, resulting in a mutant protein missing 275-298 amino acids.

[0067] Therefore, this invention provides a gene editing method, which can be optionally applied in vivo or in vitro to repair or reverse the expression of cells carrying disease-causing genes. The method of this invention offers a promising therapeutic strategy by integrating transgenes capable of supplementing disease mutations into safe harbor or native sites, providing stable and sustained gene expression for therapeutic applications. For example, in X-SCID, by integrating normal IL2RG cDNA, immune function deficiencies caused by mutations can be supplemented. In LAL-D, by repairing or replacing the mutated LIPA gene, the normal function of lysosomal acid lipase can be restored, reducing lipid accumulation and thus improving symptoms. In the examples, the inventors quantitatively analyzed the expression level of the LIPA gene using RT-qPCR, and the results showed that cells edited using the CRISPR enIE system could partially restore normal gene expression, further demonstrating the effectiveness of the system in gene therapy. Therefore, the method of this invention provides an innovative solution for gene therapy of hereditary diseases, effectively repairing or reversing the expression of cells carrying disease-causing genes, both in vivo and in vitro, with broad application prospects and clinical value.

[0068] For ease of understanding, the gene editing compositions that can be used in this invention are described in detail below:

[0069] According to embodiments of this application, by employing this composition, a novel mechanism can be used to alter the preference of cellular single-strand break (SSB) repair pathways through a gene editing system based on the nicking enzyme activity of single-strand cutting. This can improve homologous recombination / homologous repair efficiency by increasing the concentration of target nucleic acid molecules containing homologous sequences at a single DNA nick site. This can effectively achieve gene editing at specific locations and effectively avoid the adverse consequences caused by DNA double-strand cutting. At the same time, it effectively solves the problem that single-strand breaks (SSBs) are quickly repaired back to an unedited state, making it difficult to apply to gene editing alone.

[0070] Unless otherwise specified herein, the term "gene editing" as used herein refers to an operation that modifies the genetic information present in the genome. Such gene editing can be performed by manipulating genomic DNA, thereby resulting in modification of the genetic information. According to embodiments of this application, the gene editing process can be performed in vivo or in vitro.

[0071] In this document, the term "nickase" is also referred to as "nicking enzyme" or "nickase," which can generate single-stranded nicks in DNA at specific sites in the genome, without simultaneously cleaving the DNA double strand. This avoids adverse events caused by DNA double-strand breaks (DSBs), such as high-frequency base deletions or insertions, translocations, p53 pathway activation, and increased risk of tumorigenesis. According to embodiments of this application, the nickase includes at least one of the following: CRISPR-based nickase variants, TALE or ZF nickases that cleave single-stranded DNA, homing endonucleases that cleave only one strand of DNA (Meganuclease, LAGLIDADG homing endonucleases), MagaTAL, ARCUS, or a combination of the above nickase variants with a homologous recombination repair factor. Optionally, the CRISPR-based nickase includes SpCas9 (D10A), SpCas9 (H840A), and SaCas9, SpGCas9, and SpRY. The nicking enzyme variants of Cas9, ScCas9, Cas-CLOVER, Cas12, Cas13, CasX, CasMINI, IscB, and TnpB are optional. Alternatively, the CRISPR protein, TALE, or ZF nicking enzyme that cleaves single-stranded DNA includes CRISPR, TALE, and ZF variants based on MutH, FokI, I-AniI, I-OnuI, and I-TevI. Optionally, the nicking enzyme includes a fusion protein of the above nicking enzyme variants and a homologous recombination repair factor. Proteins that promote homologous repair include Rad51, Rad51 (A190L A192L), and MRN. Complex (MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51 (S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 protein or variants thereof, optionally, the nick enzyme has the protein / amino acid sequence shown in SEQ ID NO: 2.

[0072] Current gene editing methods largely rely on existing intracellular gene repair mechanisms. Referring to Figure 1, DNA repair mechanisms can be categorized into single-strand break (SSB) repair and double-strand break (DSB) repair based on the type of damage. These two methods differ fundamentally in their generation, repair mechanism, speed, and repair outcome. It is well-known in the field that single-strand DNA breaks are very common in normal cells, occurring tens of thousands of times per cell per day. Single-strand breaks (SSBs) are quickly and perfectly repaired, making it difficult to effectively initiate homologous recombination. Therefore, in the field of gene editing, greater attention is paid to improving the efficiency of homologous recombination induced by double-strand breaks (DSBs).

[0073] However, the inventors of this application believe that DSB involves the simultaneous breakage of both strands of DNA, which is a more severe form of DNA damage. Because both strands are broken, repair cannot be achieved using complementary strands. Cells typically require non-homologous end joining (NHEJ) and homologous recombination repair for repair. Inaccurate NHEJ is the primary repair pathway, and it carries a very high probability of causing mutations. Therefore, in the field of gene editing, it is generally accepted that there is greater focus on in-depth research into DSB-based gene editing to improve the homology efficiency induced by double-strand breaks (currently, many widely used gene editing methods are DSB-based programmable gene editing, including using Cas9 nuclease to generate site-specific double-strand breaks, and then using the homologous recombination-mediated DNA repair mechanism induced by double-strand breaks to knock genes into specific sites). However, the inventors of this application believe that the safety issues of gene editing based on DSB repair are very serious problems that are difficult to overcome by the DSB pathway itself. For example, NHEJ and other imprecise DNA repair pathways, which are more likely to occur after DNA double-strand breaks, often lead to adverse consequences, such as the generation of base insertion or deletion mutations, chromosomal translocations, activation of the p53 pathway, and an increased risk of tumorigenesis.

[0074] DNA single-strand breaks (SSBs) affect only one strand of a DNA double helix, leaving the other strand intact. Therefore, cells can use the undamaged strand as a template for repair, which is relatively simple, rapid, and efficient. Because the repair process uses a complete DNA strand as a template, SSB repair is usually very precise and does not introduce mutations. Therefore, the inventors of this application believe that although SSB-based gene editing is difficult to implement in practice due to the rapid and perfect repair of SSBs, it still has good application prospects because SSB repair is usually very precise and does not introduce mutations. Even if a target nucleic acid molecule containing a homologous sequence containing a target site is inserted into the genome without homologous recombination / homologous repair, it will not have a significant negative impact on the cell itself. However, as mentioned above, unlike double-strand breaks, single-strand breaks are repaired rapidly and precisely, and therefore cannot effectively generate mutations or initiate homologous recombination / homologous repair, making it difficult to apply alone to gene editing. To date, there is still no method for efficient, programmable, large-fragment editing using a single single-strand break. Paired nickases can improve gene editing efficiency by creating a gap at a close position on each of the two DNA strands, but they still involve DNA double-strand breaks. Furthermore, editing methods combining nickases and integrases often involve breaks on each of the two DNA strands, also carrying the risk of single-strand breaks (DSB). Therefore, current editing methods based on single-strand breaks require combining nickases with different operases to achieve editing. For example, base editing (BE) combines a nickase with a DNA deaminase for single-base editing, while prime editing (PE) combines a nickase with a reverse transcriptase (RT) for small fragment editing, and only small fragments (no more than 200 base pairs) can be inserted. These methods often introduce other operases besides the nickase, which can cause off-target editing byproducts, affecting the accuracy of the editing. Currently, there is no safe and efficient programmable large-fragment gene insertion method that relies solely on a single DNA gap.

[0075] Through in-depth exploration of DNA single-strand repair mechanisms, the inventors of this application discovered a novel molecular mechanism that alters the preference of DNA single-strand break (SSB) repair pathways to promote homologous recombination / homologous repair dependent on homologous sequences. Specifically, increasing the concentration of target nucleic acid molecules containing homologous sequences near DNA single-strand breaks alters the preference of individual DNA gap repair pathways, significantly increasing the proportion of single-strand DNA gaps repaired through homologous recombination / homologous repair pathways. Based on this discovery, a novel, highly efficient, and low-risk gene editing method suitable for large DNA fragment insertion was developed. This method effectively avoids the problem of low gene insertion efficiency caused by the rapid repair of single-strand breaks (SSBs) back to an unedited state, making it difficult to apply independently to gene editing.

[0076] The gene editing method of this invention can significantly improve editing efficiency and safety. According to embodiments of this application, the large DNA fragment insertion efficiency is significantly improved by using this method, achieving the highest efficiency among currently reported methods using nicks to insert large fragments at single-stranded nicks in DNA. In some sites, the editing efficiency even surpasses that of Cas9-based and double-strand break-based editing methods. In contrast, previously reported methods using nicks to first insert an integrase recognition sequence at a designated site via prime editing before inserting a large DNA fragment using the integrase have lower efficiency and create single-stranded nicks on both strands of the DNA, posing a risk of inducing double-strand breaks. Furthermore, gene editing relying solely on a single nick at a single nick without additional reverse transcriptase has not been previously reported for efficient large DNA fragment insertion, resulting in lower efficiency; it has only been used for single-base mutations or small DNA fragment insertion.

[0077] In terms of safety, the gene editing system based on a single DNA single-strand cut, as described in this application, can effectively avoid adverse consequences caused by DNA double-strand cutting, such as additional chromosomal translocations and inversions caused by DNA double-strand breaks. Furthermore, compared to Cas9 DNA double-strand cutting-mediated gene editing systems, the gene editing system of this application can significantly reduce the probability of deletions or insertions (indels) caused by editing, and significantly increase the ratio of the intended edited product (i.e., the precise insertion of a foreign gene at a designated site according to the target nucleic acid molecule) to the non-intentional edited product (gene mutation, deletion, or translocation at a designated site, without precise insertion of a foreign gene). According to a specific embodiment of this application, the ratio of intended edited products to non-intentional edited products can be increased to over 16, and the probability of non-intentional edited products can be reduced to 0.5%. In contrast, the ratio of intended to non-intentional edits in Cas9-based gene editing is often less than 1, and the probability of non-intentional edits is often higher than 50%. Compared to Cas9-based gene editing, the probability of indels caused by the gene editing system of this application can be reduced to below 1 / 217.

[0078] Because the gene editing method of this invention causes only a single DNA strand break, it avoids the adverse consequences of double-strand DNA breaks. This enables highly efficient and low-risk simultaneous and repeated editing of multiple gene sites, which is difficult to achieve with traditional gene editing methods based on double-strand DNA breaks. This further improves efficiency and creates the possibility for multiple treatments in disease management.

[0079] According to embodiments of this application, in conventional gene editing methods based on double-strand DNA breaks, almost all sites not containing the target insertion edit mutate after the initial insertion edit on the cell. However, in cells edited using the gene editing method of this invention, over 99% of the sites not containing the target insertion edit retain the wild type. Therefore, the overall editing efficiency can be improved through repeated editing. This repeatable editing characteristic is suitable for the practical needs of multiple-dose treatment in disease therapy, overcoming the limitations of previous gene editing methods based on double-strand DNA breaks.

[0080] According to embodiments of this application, traditional gene editing methods based on double-strand DNA breaks can detect translocation products after simultaneously editing two sites located on two separate chromosomes, increasing the risk of gene mutations and even cell carcinogenesis. However, the gene editing method of this application does not detect translocation products after performing the same operation, allowing for safe and efficient simultaneous gene insertion at multiple sites. This aligns with the needs of genetic disease treatment and simultaneous multi-site editing in engineered cells.

[0081] These characteristics demonstrate the unique advantages of the present invention's highly efficient and low-risk gene editing method based on a single DNA single-strand cut (SSB) over traditional gene editing methods based on double-strand DNA breaks (DSB).

[0082] Referring to Figures 2, 3, and 4, according to embodiments of this application, the inventors utilize a programmable nicking enzyme complex (which may include a protein complex containing gRNA) to recruit DNA donors with bilateral homologous arms. The nicking enzyme cleaves DNA at the target site, creating a single-stranded gap without producing double-strand breaks. Simultaneously, it recruits DNA donors (dsDNA or ssDNA) containing homologous sequences to the gap as templates to promote DNA gap repair via homologous recombination / homologous repair pathways. This improves the efficiency of precise DNA insertion at the target site via homologous recombination while avoiding the risks associated with DSB (Double Straining), thus enhancing the safety of gene editing. Specifically, the nicking enzyme complex is configured to connect with a first recruitment component, and the target nucleic acid molecule is configured to connect with or bind to a second recruitment component. The first and second recruitment components are adapted to form specific bindings. Therefore, through the combined action of the first and second recruitment components, target nucleic acid molecules (DNA donors) containing homologous sequences with target sites can be recruited to the nicks formed by the nick enzyme, thereby improving the efficiency of precise DNA insertion at the target site via homologous recombination / homologous repair pathways, while avoiding the risks caused by DSB and improving the safety of gene editing.

[0083] According to embodiments of this application, the target nucleic acid molecule used herein, also referred to as a "DNA donor" or "template DNA," refers to a foreign DNA sequence that provides the required genetic information during gene editing or gene transfer. These sequences contain DNA fragments at both ends homologous to specific regions in the target cell's genome (i.e., homologous arms), and the genetic information to be inserted, replaced, or repaired. When inserting large fragments (greater than 500 bp), the homologous arm length is 100-1500 bp; when performing base substitution, repair, or small fragment insertion, the homologous arm can be as short as 15-50 bp. The insertion site of the foreign DNA, determined by the homologous arms, is within 20 bp of the nick site. DNA donors play a crucial role in gene therapy, genetic engineering, and biotechnology research. They can serve as templates for homologous repair, used by cells to repair damage caused by DNA breaks, and in this process, integrate the genetic information from the donor DNA into the genome at the target site. According to embodiments of this application, the target nucleic acid molecule may include an insert fragment and homologous arms located on both sides of the insert fragment, wherein the length of the insert fragment is not less than 100 bp, for example, not less than 200 bp, for example, not less than 300 bp, for example, not less than 500 bp, for example, not less than 1 kb. According to embodiments of this application, the inventors, using the method of this application, can even successfully insert DNA fragments of up to at least 10 kb into the target site. The length of the homologous arms is determined based on the length of the insert fragment.

[0084] According to embodiments of this application, a recruitment component refers to a component used to recruit specific proteins or other molecules, thereby promoting specific intermolecular interactions. Specifically, a first recruitment component and a second recruitment component are designed to bind to a nicking enzyme complex and a target nucleic acid molecule, respectively. This binding promotes the recruitment of target nucleic acid molecules containing homologous sequences to the nick formed by the nicking enzyme. In embodiments of this application, the first recruitment component refers to a molecule linked to the nicking enzyme complex, and the second recruitment component refers to a molecule linked to or bound to the target nucleic acid molecule, thereby promoting specific binding between the first and second recruitment components. This ensures that the nicking enzyme complex can accurately recruit target nucleic acid molecules containing homologous sequences of the target site to the single-strand nick, thereby achieving gene editing through homology-directed repair. This improves the efficiency of precise DNA insertion at the target site via homology recombination / homology repair pathways, while avoiding the risks caused by DSB (Dual Straining of DNA) and improving the safety of gene editing.

[0085] According to embodiments of this application, the recruitment component may be a protein, peptide, small molecule, oligonucleotide, or other type of molecule. According to embodiments of this application, the first recruitment component and the second recruitment component are adapted to form a specific binding through at least one of the following combinations: a nucleic acid fragment and a protein that specifically recognizes the nucleic acid fragment; a protein tag and a ligand, nucleic acid aptamer, or binding protein of the protein tag; an antigen and an antibody; optionally modified non-natural amino acids and corresponding binding ligands; optionally modified non-natural bases and corresponding binding ligands; a sequence-specific DNA / RNA binding protein and its specific binding sequence; a structure-specific DNA / RNA binding protein and its specific binding sequence; a specific nucleic acid modification and a corresponding binding protein, antibody, or ligand of the nucleic acid modification; a viral protein or a functional fragment thereof and a corresponding cellular or soluble viral receptor; a viral protein and its specific antibody; a viral envelope protein and a target cell membrane lipid / protein component; a viral protein and a DNA / RNA sequence recognized by the viral protein; a small molecule and a protein or nucleic acid sequence that specifically recognizes the molecule.

[0086] Specifically, according to embodiments of this application, the first recruitment component and the second recruitment component are adapted to form a specific binding through at least one of the following combinations: Rep protein-PCV recognition sequence from porcine circovirus 2PCV; TALE and its specific binding sequence; ZF and its specific binding sequence; CRISPR protein and variants and their specific recognition sequences; ParB-parS; LacI-lacO; TetR-tetO; CymR-cuO; non-natural amino acids containing azido groups and ligands containing alkyne groups; non-natural amino acids containing azido groups and non-natural bases containing alkyne groups; non-natural amino acids containing cyclopropene groups and tetrazine compounds; non-natural amino acids containing trans-cyclooctene (TCO) and S-tetrazine (Tetrazine). e) Non-natural amino acids containing azide groups with cyclopropane-cyclooctylene; non-natural amino acids containing tetrazine groups with cyclopropene or trans-cyclooctene; AeF-DBCO; AzF-DBCO; NAEK-DBCO; HaloTag-chlorinated alkane ligand; SnapTag-corresponding ligand; TMPTag-corresponding ligand; monomeric or non-monomeric or tandem streptavidin-biotin; viral DNA / RNA binding proteins-their binding sequences or DNA / RNA components; viral or cell-derived DBP, SV40, NS1, ICP8, ORF, E1, E2, integratese, UL9, HMG, Rep, NS1, IE175, NP, N, NS, GAG, RRM, HLH, AT-hook motif, EBNA1 protein-their protein recognition sequences or components; SunTag-anti-GCN4 single-chain variable sequence antibody; MoonTag-gp41 nanobody.

[0087] According to embodiments of this application, the first recruitment component and the nicking enzyme are covalently linked. For example, the first recruitment component and the nicking enzyme constitute a fusion protein. According to specific embodiments of this application, optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; optionally, the first recruitment component includes at least one TALE or ZF protein, and the second recruitment component is a TALE or ZF binding sequence; optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; optionally, the... The first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; optionally, the first recruitment component includes at least one Rep protein, and the second recruitment component is a Rep protein recognition sequence; optionally, the first recruitment component includes at least one monomeric or non-monomeric streptavidin or biotin antibody, and the second recruitment component is biotin; optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; optionally, the first recruitment component may include a tandem combination of multiple monomers. Therefore, the efficiency of recruiting target nucleic acid molecules (donor DNA) to the nick for homology repair can be further improved.

[0088] According to embodiments of this application, the first recruitment component and the nicking enzyme constitute a fusion protein. Optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; optionally, the first recruitment component includes at least one TALE or ZF protein, and the second recruitment component is a TALE or ZF binding sequence; optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence. R sequence; Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; Optionally, the first recruitment component includes at least one Rep protein, and the second recruitment component is a Rep protein recognition sequence; Optionally, the first recruitment component includes at least one monomeric or non-monomeric streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; Optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; Optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; Optionally, the first recruitment component includes at least two monomers in tandem.

[0089] According to an embodiment of this application, the combination of the first recruitment component and the second recruitment component constitutes a fusion protein.

[0090] According to an embodiment of this application, the first recruitment component and the nick enzyme are connected by a linker.

[0091] According to embodiments of this application, the linker has 15-150 amino acids, for example, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids, or 16-150 or 17-150 amino acids. According to specific embodiments of this application, the linker has the amino acid sequence shown in SEQ ID NO: 1 or has at least 50% homology with SEQ ID NO: 1, for example, at least 80% homology, at least 90% homology, or at least 95% homology. According to embodiments of this application, the linker is a polypeptide chain with a certain degree of flexibility and elasticity. By employing a linker, the nick enzyme is allowed to maintain its connection with the recruitment component while possessing a certain degree of flexibility, thereby interacting more effectively with the target nucleic acid molecule. According to embodiments of this application, the use of linkers can reduce the spatial obstacles that rigid structures may cause, allowing nicking enzymes to recruit target nucleic acids more freely. In addition, the design of using linkers can improve the stability and functionality of the complex, thereby improving the accuracy and efficiency of gene editing, reducing non-specific binding and off-target effects, and thus playing an important role in gene therapy and biomedical research.

[0092] SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 1).

[0093] According to some embodiments of this application, the first recruitment component is non-covalently linked to the nicking enzyme. For example, the nicking enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment.

[0094] According to specific embodiments of this application, optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; optionally, the first recruitment component includes at least one TALE or ZF protein, and the second recruitment component is a TALE or ZF binding sequence; optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; optional Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; optionally, the first recruitment component includes at least one Rep protein, and the second recruitment component is a Rep protein recognition sequence; optionally, the first recruitment component includes at least one monomeric streptavidin, and the second recruitment component is biotin; optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; optionally, the first recruitment component includes at least two tandemly connected components comprising the above elements.

[0095] According to a specific embodiment of this application, the nick enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment.

[0096] According to specific embodiments of this application, the protein tag binding fragment includes at least one of the following types that specifically recognize the protein tag:

[0097] scFv, Fab, Fab', F(ab')2, Fv, Nanobody, Bispecific antibody, Minibodies, Single-domain antibody, The protein tag binding protein.

[0098] According to specific embodiments of this application, the protein tag is at least one of the GCN4 sequence and the gp41 sequence.

[0099] According to an embodiment of this application, the applicant proposes a scheme to amplify the recruitment effect. Referring to Figure 5, it shows a schematic diagram of recruiting multiple target nucleic acids (donors) to nick sites through an amplification mechanism to improve homology repair efficiency.

[0100] According to specific embodiments of this application, the guiding nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases to directly or indirectly bind multiple first recruitment components;

[0101] According to embodiments of this application, the first recruitment component is directly or indirectly linked to one or more binding proteins of the nucleic acid aptamers or binding proteins of non-natural bases.

[0102] According to embodiments of this application, the guiding nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases, such as at least one, at least two, or 3 to 100 of the nucleic acid aptamers or non-natural bases.

[0103] According to specific embodiments of this application, the combination of nucleic acid aptamers and binding proteins includes at least one selected from the following:

[0104] MS2-MCP, PP7-PCP, BoxB-lambdaN, Com-com, Streptavidin aptamer-streptavidin.

[0105] Optionally, the first recruitment component may be directly or indirectly linked to a binding protein of a non-natural base.

[0106] According to a specific embodiment of this application, the guiding nucleic acid molecule carries at least one nucleic acid aptamer (such as MS2, PP7, lambdaN, com) or non-natural bases, the first recruitment element (such as TALE, ZF, Rep protein, or monomeric streptavidin) is fused with a protein that specifically recognizes the nucleic acid aptamer (such as MCP, PCP, BoxB, Com), and the target nucleic acid molecule has a second recruitment element (such as a TALE-specific binding sequence, a Rep binding sequence, or biotin), and the first recruitment element specifically binds to the second recruitment element.

[0107] According to specific embodiments of this application, the guide nucleic acid molecule carries multiple MS2 or PP7, for example, at least one, at least two, or 3 to 100 of the nucleic acid aptamers or non-natural bases.

[0108] According to a specific embodiment of this application, the binding protein of the nucleic acid aptamer binds to a protein tag, the first recruitment component is linked to at least one protein tag binding fragment, and the protein tag binding protein is adapted to specifically bind to the protein tag.

[0109] According to a specific embodiment of this application, the nick enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment.

[0110] According to a specific embodiment of this application, the first recruitment component is directly or indirectly linked to a plurality of proteins that specifically recognize the protein tag.

[0111] According to a specific embodiment of this application, the nick enzyme is fused with at least one protein tag to directly or indirectly bind to multiple first recruitment components;

[0112] According to a specific embodiment of this application, the first recruitment component is fused with a protein that specifically recognizes the protein tag.

[0113] According to a specific embodiment of this application, the nick enzyme is fused with at least one GCN4 sequence and / or gp41 sequence, the first recruitment element (such as TALE or ZF, Rep protein, monomeric streptavidin) is fused with an scFv or scFV variant that specifically recognizes the GCN4 sequence and / or gp41 sequence, the target nucleic acid molecule has a second recruitment element (such as a TALE or ZF specific binding sequence, a Rep binding sequence, biotin), and the first recruitment element specifically binds to the second recruitment element.

[0114] According to specific embodiments of this application, the nicking enzyme is fused with a plurality of the protein tags, for example, at least one, at least two, or 3 to 30 of the protein tags.

[0115] According to a specific embodiment of this application, the scFv or scFV variant of the GCN4 sequence and / or gp41 sequence is specifically identified and fused with a plurality of tandem first recruitment elements, such as at least one, at least two, or 2 to 30 of the first recruitment elements.

[0116] According to embodiments of this application, multiple target nucleic acid molecules containing recruitment components can also be recruited by adding multiple nucleic acid aptamers or non-natural bases to the guide nucleic acid molecule to recruit proteins or ligands that specifically bind to it. This allows for the recruitment of multiple target nucleic acid molecules to a nick site (also known as a "notch"), thereby improving the efficiency of gene editing through homologous recombination of target nucleic acids at the nick site.

[0117] According to the embodiments of this application, the gene editing methods employed are not particularly limited. They can be techniques such as CRISPR-Cas9 with directed nucleic acid molecules, or they can be TALE nickase (Transcription Activator-Like Effector nickase) and ZF nickase (Zinc Finger Nickase) without directed nucleic acid molecules. TALE nickase is a nickase variant that cuts only one DNA strand, based on a transcription activator-like effector. Its core technology consists of tandem TALE (transcription activator-like effector) protein repeat sequences, each repeat consisting of a TALE protein of 33-35 amino acids in length, with each TALE protein specifically recognizing one base. In addition, TALE nickase also contains nuclear localization sequences and nucleases such as FokI, MutH, and I-TevI, or their nickase variants. Changing the TALE protein sequence can alter the recognition site. ZF Nickase is similar, being a nickase variant based on zinc-finger nuclease (ZFN) technology. Its core is a tandem zinc-finger (ZF) structure, with each zinc finger specifically recognizing three bases. ZF Nickase also includes NLS and nucleases that cleave only one DNA molecule. The recognition and cleavage sites of ZF Nickase can be altered by designing the tandem sequence of the zinc finger structure. The nickase can also be a fusion protein of the above nickase variants and homologous recombination repair factors. Proteins that promote homologous repair include Rad51, Rad51(A190LA192L), MRN Complex (MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51(S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 proteins, or variants thereof.

[0118] According to embodiments of this application, the nick enzyme complex further includes an optional guide nucleic acid molecule that can direct the nick enzyme to a predetermined genomic site for targeted gene editing.

[0119] According to embodiments of this application, the guided nucleic acid molecule can carry multiple nucleic acid aptamers or non-natural bases.

[0120] According to embodiments of this application, the first recruitment component is directly or indirectly linked to the binding proteins of the plurality of nucleic acid aptamers. Those skilled in the art will understand that the connection method between the first recruitment component and the binding proteins of the plurality of nucleic acid aptamers is not particularly limited; it can be a direct connection or an indirect connection. Thus, the first recruitment component can be linked to the nicking enzyme complex through the binding of the nucleic acid aptamer to its binding protein.

[0121] According to embodiments of this application, the combination of nucleic acid aptamers-binding proteins includes at least one selected from the following: MS2-MCP, PP7-PCP, BoxB-lambdaN, Com-com, and streptavidin aptamer-streptavidin.

[0122] According to embodiments of this application, the binding protein of the nucleic acid aptamer binds to a protein tag, the first recruitment component is linked to at least one protein tag binding fragment, and the protein tag binding protein is adapted to specifically bind to the protein tag. This amplifies the recruitment effect and improves the efficiency of gene editing.

[0123] According to embodiments of this application, the first recruitment component and the second recruitment component can constitute a fusion protein. Of course, those skilled in the art will understand that the fusion protein can contain multiple first or second recruitment components. The nick enzyme complex can be directly or indirectly linked to the first recruitment component, and the donor DNA can be directly or indirectly linked to the second recruitment component, thereby achieving a recruitment effect, or even an amplified recruitment effect, further improving the efficiency of gene editing.

[0124] According to embodiments of this application, the nicking enzyme includes at least one of the following: a nicking enzyme variant based on a CRISPR protein, a TALE or ZF nicking enzyme that cuts single-stranded DNA, a homing endonuclease variant that cuts only one strand of DNA, or a combination of the above nicking enzyme variants with a homologous recombination repair factor.

[0125] According to embodiments of this application, the nicking enzymes based on CRISPR proteins include SpCas9(D10A), SpCas9(H840A), and nicking enzyme variants of SaCas9, SpG Cas9, SpRY Cas9, ScCas9, Cas12, Cas13, CasX, CasMINI, IscB, and TnpB.

[0126] According to embodiments of this application, the TALE or ZF nicking enzymes that cleave single-stranded DNA include TALE and ZF variants based on MutH, FokI, I-AniI, I-OnuI, and I-TevI.

[0127] According to an embodiment of this application, the nick enzyme has the protein / amino acid sequence shown in SEQ ID NO: 2.

[0128] According to embodiments of this application, the gene editing system may further include multiple nuclear localization signals, thereby enabling the nick enzyme complex and donor DNA to better enter the cell nucleus for gene editing.

[0129] According to embodiments of this application, the nicking enzyme comprises a combination of the above-mentioned nicking enzyme variants and homologous recombination repair factors. Proteins that can promote homologous repair include Rad51, Rad51(A190L A192L), MRN Complex(MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51(S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 proteins or variants thereof.

[0130] According to embodiments of this application, the target nucleic acid molecule is plasmid DNA, viral DNA / RNA, double-stranded DNA, or single-stranded DNA.

[0131] According to embodiments of this application, the target nucleic acid molecule contains a homologous sequence, which can serve as a template for homologous recombination / homologous repair / gene editing, providing a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a designated site through homologous recombination / homologous repair, or for performing base substitution, repair, or small fragment DNA insertion, replacement, or repair at a specific site through homologous recombination / homologous repair.

[0132] According to embodiments of this application, the target nucleic acid molecule contains homologous sequences, which can serve as templates for homologous recombination / homologous repair / gene editing, providing exogenous nucleic acid sequences that provide the required genetic information. This is suitable for inserting exogenous nucleic acid sequences into designated sites through homologous recombination / homologous repair, or for inserting, replacing, or repairing large fragments at specific sites through homologous recombination / homologous repair.

[0133] According to embodiments of this application, the exogenous nucleic acid sequence has homologous arms at both ends. The homologous arms are homologous to the nucleic acid sequence of a specific region in the target genome. When the target nucleic acid molecule is used to insert a large fragment with a gene size greater than 500 bp, the length of the homologous arm is 100-1500 bp. When the target nucleic acid molecule is used for base substitution, repair, or small fragment insertion, the homologous arm can be as short as 15-50 bp.

[0134] According to an embodiment of this application, the insertion position of the exogenous nucleic acid sequence is within 100 bp of the DNA single-strand nick site.

[0135] According to embodiments of this application, the target nucleic acid molecule carries an identifiable modification or a specific protein-binding sequence.

[0136] According to embodiments of this application, the target nucleic acid molecule is prepared by plasmid or PCR.

[0137] In summary, the novel gene editing system proposed in this application innovatively uses a single nick enzyme to create a single-strand nick while recruiting at least one DNA donor containing a homologous sequence. It unexpectedly discovers a novel molecular mechanism that alters the preference for single DNA gap (single-strand break) repair pathways, making homologous recombination / homologous repair a crucial pathway for single-strand break repair. This effectively solves the problem of low gene insertion efficiency caused by the rapid repair of single-strand breaks (SSBs) back to an unedited state, making it difficult to apply alone to gene editing, and significantly improves the efficiency of gene insertion using this method. According to the embodiments of this application, by employing this gene editing system, based on the nick enzyme activity of single-strand cutting, and utilizing the cellular single-strand break (SSB) repair mechanism, gene editing at specific locations can be effectively achieved by enhancing homologous recombination / homologous repair of target nucleic acid molecules. Furthermore, it effectively avoids the adverse consequences caused by DNA double-strand cutting, and also effectively avoids the problem of difficulty in applying single-strand breaks (SSBs) to gene editing alone due to rapid repair. It was also unexpectedly discovered that, compared to traditional methods that rely on DNA double-strand breaks, this method also improved the cell viability, proliferation rate, and total number of cells after editing.

[0138] According to embodiments of this application, the gene editing system of this application enables programmable, efficient, and low-risk gene knock-in, which holds great promise for treating complex genetic diseases and cancers. It also enables safe and efficient integration of large DNA fragments. Specifically, the enhanced insertion editing (enIE) system based on a single-strand cutting programmable nickase developed by the inventors of this application provides a highly efficient, low-risk gene insertion method based on DNA nick sites and dependent on donor homologous sequences, enabling programmable large DNA fragment insertion. The CRISPR-based programmable enIE system can achieve efficient integration of large DNA fragments at different genomic sites in different cell types (including human primary T lymphocytes). Compared to Cas9, it significantly reduces the insertion or deletion mutation rate. In cell models containing pathogenic mutations, enIE restores normal gene expression by inserting therapeutic genes into safe sites or their natural sites, achieving a higher insertion rate than the Cas9 system. Furthermore, enIE can efficiently insert large fragments into primary T cells while minimizing cytotoxicity, significantly increasing the number of T cells containing the targeted insertion compared to Cas9. enIE also enables simultaneous multi-site insertion, allowing multiple fragments to be inserted into multiple different genomic sites in a single transfection, while minimizing the risk of translocation and non-target nucleic acid insertion. Additionally, according to embodiments of this application, the inventors have proposed an optimized gene editing system that can further amplify recruitment effects and improve insertion efficiency. Therefore, the enIE system, by achieving efficient, site-specific large-fragment DNA insertion, avoids the risk of aberrant editing due to double-strand breaks, improving editing efficiency and safety, making it a powerful tool for advancing personalized medicine.

[0139] According to embodiments of this application, the composition may further include a delivery system that loads the nick enzyme complex or its encoded nucleic acid molecule and the target nucleic acid molecule.

[0140] According to embodiments of this application, the composition is suitable for use in conjunction with base editing and prime editing.

[0141] According to embodiments of this application, the nucleic acid molecule encoding the nick enzyme complex is disposed on DNA or RNA.

[0142] According to embodiments of this application, the nucleic acid molecule encoding the nick enzyme complex is plasmid DNA.

[0143] According to embodiments of this application, the nucleic acid molecule encoding the nick enzyme complex is a viral vector DNA / RNA.

[0144] According to embodiments of this application, the nucleic acid molecule encoding the nick enzyme complex is RNA.

[0145] According to embodiments of this application, the nick enzyme complex is a protein or a protein-RNA complex.

[0146] According to embodiments of this application, the delivery system is suitable for liposome transfection, microinjection, electrotransfection, cationic polymer transfection, lipid nanoparticle transfection, and viral vector delivery.

[0147] According to the embodiments of this application, the mass ratio of the plasmid to the target nucleic acid molecule is (1-10):(1-10), preferably 3:1, 3:4, 1:1, 1:2, and more preferably 1:1, which can improve the working efficiency of the gene editing system in cells.

[0148] According to an embodiment of this application, the mass ratio of the RNA to the target nucleic acid molecule is (1-10):(1-10).

[0149] According to an embodiment of this application, the mass ratio of the protein or protein-RNA complex to the target nucleic acid molecule is (1-10):(1-10).

[0150] Methods for gene editing of cells

[0151] In a second aspect of this application, a method for gene editing of cells is provided. According to an embodiment of this application, the method includes: contacting the composition described in the first aspect of this application with cells to introduce a nick enzyme complex and a target nucleic acid molecule into the cells for gene editing at at least one given site of the cells.

[0152] According to the method of the embodiments of this application, based on the nicking enzyme activity of single-strand cutting, the single-strand break (SSB) repair mechanism of cells can effectively achieve gene editing at a specific location by improving homologous recombination / homologous repair of the target nucleic acid molecule. It can also effectively avoid the adverse consequences caused by DNA double-strand cutting, and effectively avoid the problem that it is difficult to apply to gene editing alone due to the rapid repair of single-strand breaks (SSB).

[0153] According to embodiments of this application, compared to traditional methods that rely on DNA double-strand breaks, the viability, proliferation rate, and total number of cells edited using this method are also improved.

[0154] According to embodiments of this application, the given sites that can be used include: safe harbor sites, cell engineering editing sites, and disease gene sites.

[0155] According to embodiments of this application, the contact is performed in vitro within cells or in vivo.

[0156] According to embodiments of this application, the cells are derived from a patient or allogeneic organism, and are introduced into the patient's body after contact with the composition described in the first aspect of this application.

[0157] According to embodiments of this application, the given site includes at least one of the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3 alpha (DNMT3A), Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-b receptor II, protein tyrosine phosphatase 1B (PTP1B), CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein(PRDM1), CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC 2. WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1 and TIGER.

[0158] According to embodiments of this application, the gene editing includes introducing a point mutation, gene insertion, deletion, or substitution at at least one of the given sites.

[0159] According to embodiments of this application, the gene editing includes inserting a nucleic acid fragment of not less than 100 bp, for example, not less than 1 kb, at at least one given site; when the given site inserts a large fragment of gene size greater than 500 bp, the homologous arm length is 100-1500 bp; when base substitution, repair, or small fragment DNA insertion is performed at the given site, the homologous arm may also be 15-1000 bp.

[0160] According to embodiments of this application, the gene editing system of this application can effectively and safely insert large fragments of DNA.

[0161] According to the embodiments of this application, the gene editing system of this application can not only insert short DNA fragments, but also safely and efficiently insert long nucleic acid fragments, such as nucleic acid fragments not less than 1kb. Therefore, it can be effectively applied to the genome modification of cells, such as for constructing recombinant immune cells. It should be noted that when inserting a large fragment with a gene size greater than 500bp at the given site, the length of the homologous arm can be 100-1500bp. When performing base substitution, repair, or inserting small DNA fragments at the given site, the length of the homologous arm can also be 15-1000bp.

[0162] According to the embodiments of this application, since the cell genome can be safely and effectively edited using the method of this application, and the adverse consequences caused by DNA double-strand cutting can be effectively avoided, the cell genome can be edited multiple times, as shown in Figure 6.

[0163] According to embodiments of this application, the method may further include: performing at least one repeated editing on the gene-edited cells, wherein the repeated editing targets the same or different given sites for the same or different gene editing.

[0164] Therefore, according to embodiments of this application, gene editing may include simultaneously introducing point mutations, gene insertions, deletions, or substitutions at multiple given sites without causing adverse events such as additional chromosomal translocations or inversions caused by DNA double-strand cutting.

[0165] According to embodiments of this application, the plurality of donation sites are located on at least two different chromosomes.

[0166] According to embodiments of this application, the proportion of non-target indels also decreases significantly.

[0167] It should be noted that the description used in this application, "no adverse consequences caused by DNA double-strand cutting, including additional chromosomal translocations and inversions," should be understood as meaning that the gene editing system of this application significantly reduces the probability of adverse consequences caused by DNA double-strand cutting compared to Cas9-based double-strand break-based editing systems. For example, when editing with a Cas9-based double-strand cutting system, the translocated or inverted target bands can be detected by PCR detection of potential translocation or inversion sites; however, the gene editing system of this application cannot detect translocated or inverted target bands. Furthermore, compared to Cas9-based double-strand cutting systems, the gene editing system of this application also significantly reduces the probability of deletion or insertion mutations (indels) and significantly increases the ratio of target edited products to non-target edited products. For example, the ratio of target edited products to non-target edited products can be increased to over 16, and the probability of non-target edited products can be reduced to below 0.5%. In comparison, the ratio of targeted to non-targeted editing in Cas9-based gene editing is typically less than 1, with the probability of non-targeted editing generally exceeding 50%. Compared to traditional Cas9-based gene editing, according to embodiments of this application, the probability of indels caused by the gene editing system of this application can be reduced to below 1 / 217. Compared to paired nickases-based gene editing, according to embodiments of this application, the probability of indels caused by the gene editing system of this application is also reduced to below 1 / 49.8. It should be noted that those skilled in the art will understand that the actual proportion of indels generated through gene editing will be further lower than the proportions described above. Therefore, these proportions are the results obtained after gene sequencing following gene editing. Since conventional detection methods cannot distinguish between indels caused by editing and indels generated by spontaneous cell mutations, the detected proportions will include single nucleotide polymorphisms (SNPs) and indels generated by spontaneous mutations. The actual proportion of indels generated through gene editing will be lower than the detected proportions. Furthermore, the proportion of indels detected by different detection methods may vary.

[0168] Methods for long-fragment gene editing in cells

[0169] In a third aspect of this application, a method for long-fragment gene editing of cells is proposed. According to an embodiment of this application, the method includes: contacting the composition described in the first aspect of the invention with cells to introduce a nick enzyme complex and a target nucleic acid molecule into the cells, wherein the target nucleic acid molecule includes an insert fragment and homologous arms located flanking the insert fragment, wherein the length of the insert fragment is not less than 100 bp, for example not less than 200 bp, for example not less than 300 bp, for example not less than 500 bp, for example not less than 1 kb, and can even be as high as at least 10 kb.

[0170] According to embodiments of this application, the length of the homologous arm is determined based on the length of the inserted fragment. Typically, the length of the homologous arm is positively correlated with the length of the inserted fragment. In practice, those skilled in the art can also verify and optimize this through experiments to obtain a homologous arm length that achieves the desired effect.

[0171] For example, according to the embodiments of this application, when a large fragment larger than 500 bp is inserted at the given site, the length of the homologous arm can be 100-1500 bp. When base substitution, repair or small fragment insertion is performed at the given site, the length of the homologous arm can also be 15-1000 bp.

[0172] The inventors of this application unexpectedly discovered that, based on the unique working mechanism of the gene editing system of this application, long-fragment gene insertion into the cellular genome can be effectively achieved without adverse events caused by DNA double-strand cutting, such as additional chromosomal translocations or inversions. Simultaneously, it promotes the intranuclear delivery of target nucleic acid molecules and reduces adverse effects such as intracytoplasmic aggregation. Compared to traditional methods relying on DNA double-strand breaks, the viability, proliferation rate, and total quantity of edited cells are also improved. According to embodiments of this application, the multiple donor sites are located on at least two different chromosomes. According to embodiments of this application, the proportion of non-target indels is also significantly reduced. According to embodiments of this application, the viability, proliferation rate, and yield of edited cells are also improved. Therefore, the application fields of gene editing can be greatly expanded, such as the treatment of genetic diseases and the construction of recombinant immune cells such as CAR-T cells.

[0173] Methods of gene therapy for patients

[0174] In a fourth aspect of this application, a method for gene therapy on a patient is proposed. According to embodiments of this application, the method includes: determining the sequences of a target nucleic acid molecule and a guide nucleic acid molecule based on the patient's target gene; contacting the composition described in the first aspect of this application with cells from the patient, or introducing an enzyme encoding a nick enzyme complex and a target nucleic acid molecule into the cells via the methods described in the second and third aspects of this application, to perform gene editing at at least one given site in the cells. The method according to embodiments of this application can effectively perform targeted repair on the patient's cells, thereby improving the efficiency of disease treatment.

[0175] According to embodiments of this application, a method for treating a patient may further include: performing at least one repeated editing on the gene-edited cells, wherein the repeated editing targets the same or different given sites for the same or different gene editing. As mentioned above, according to embodiments of this application, since the method of this application can efficiently and with low risk perform gene editing on the cell genome and can effectively avoid the adverse consequences caused by DNA double-strand cutting, the cell genome can be edited multiple times. For example, referring to Figure 6, according to embodiments of this application, the method may further include: performing at least one repeated editing on the gene-edited cells, wherein the repeated editing targets the same or different given sites for the same or different gene editing. Thus, according to embodiments of this application, gene editing may include simultaneously introducing point mutations, gene insertions, deletions, or substitutions at multiple given sites without causing adverse consequences caused by DNA double-strand cutting, including additional chromosomal translocations and inversions.

[0176] According to embodiments of this application, the plurality of donation sites are located on at least two different chromosomes.

[0177] According to embodiments of this application, the cells are derived from a patient or allogeneic organism, and are introduced into the patient's body after contact with the composition described in the first aspect of this application.

[0178] According to embodiments of this application, the contact is performed in vivo or in vitro.

[0179] According to an embodiment of this application, the patient suffers from a gene mutation-related disease.

[0180] According to an embodiment of this application, the patient's cells are non-germinal cells.

[0181] According to embodiments of this application, the given site is a disease-related gene, a cell engineering editing site, or a safe harbor site.

[0182] According to embodiments of this application, the cells include patient-derived or allogeneic stem cells, somatic cells, cancer cells, and immune cells.

[0183] According to embodiments of this application, the given site includes at least one of the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3 alpha, Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-β receptor II, protein tyrosine phosphatase 1B, CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein, CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC 2. WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1 and TIGER.

[0184] According to embodiments of this application, the gene mutation-related diseases include hematologic diseases, autoimmune diseases, deafness, eye diseases, metabolic diseases, AIDS, musculoskeletal diseases, lymphoma, B-cell leukemia and multiple myeloma, bladder cancer, liver cancer, lung cancer, epithelial cancer, colon cancer, cervical cancer, and other cancers; hematologic diseases such as sickle cell anemia and thalassemia; eye diseases such as hereditary retinal diseases; musculoskeletal diseases such as Duchenne muscular dystrophy, ALS (amyotrophic lateral sclerosis), and myotonic dystrophy type 1 / II; metabolic diseases such as lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, and hereditary tyrosinemia type 1; and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, systemic vasculitis, and type 1 diabetes.

[0185] It should be noted that the above-mentioned treatment methods can be gene therapy, cell therapy, or a combination of both. The targeted cells are non-germinal cells and can be derived from the patient's own body or from an allogeneic source. Cell therapy involves introducing autologous or allogeneic stem cells, cancer cells, or immune cells into the patient's body, endowing the cells with new capabilities to treat or prevent disease. For example, stem cell therapy utilizes the self-renewal and differentiation capabilities of stem cells to repair or replace damaged tissue. By adding additional genes to stem cells, especially gene manipulation targeting disease-related genes or safe harbor sites, their therapeutic potential can be further enhanced. For example, by improving their ability to differentiate into specific cell types or enhancing their immunomodulatory function; or by modifying immune cells using endogenous gene regulatory mechanisms to express immunomodulatory factors and other genes that enhance anti-tumor responses at tumor sites; or, for example, CAR-T cell therapy involves collecting the patient's T cells, introducing the CAR gene through gene transfer technology, and then re-injecting these engineered cells into the patient to enhance their immune response against specific cancer cells, thereby better exerting their therapeutic effect.

[0186] use

[0187] In a fifth aspect of this application, the use of the composition described in the first aspect of this application or gene-edited cells obtained by the methods of the second and third aspects of this application in the preparation of a medicament for treating and / or preventing a disease. According to embodiments of this application, the disease includes at least one of gene mutation-related diseases and autoimmune diseases. According to embodiments of this application, the use of the composition described in the first aspect of this application or gene-edited cells obtained by the methods of the second and third aspects of this application can effectively target and repair the patient's cells, thereby improving the efficiency of disease treatment.

[0188] According to embodiments of this application, the disease is a single-gene disease or a polygenic disease.

[0189] According to embodiments of this application, the diseases include cancer, hematologic disorders, autoimmune diseases, deafness, eye diseases, metabolic diseases, AIDS, and musculoskeletal disorders. Optionally, they include sickle cell anemia and thalassemia, hereditary retinal diseases, Duchenne muscular dystrophy, ALS (amyotrophic lateral sclerosis), myotonic dystrophy type I / II, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type I, lymphoma, autoimmune diseases, B-cell leukemia, multiple myeloma, bladder cancer, liver cancer, lung cancer, epithelial cancer, colon cancer, cervical cancer, severe combined immunodeficiency, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type I, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, systemic vasculitis, and type 1 diabetes.

[0190] Methods for constructing gene-edited organisms

[0191] In a sixth aspect of this application, a method for constructing a gene-edited organism is provided. According to embodiments of this application, the method includes: contacting a composition described in the first aspect of this application with a cell, or introducing into the cell an enzyme encoding a nick enzyme complex and a target nucleic acid molecule using the methods described in the second and third aspects of this application, to perform gene editing at at least one given site in the cell, and obtaining the gene-edited organism based on the edited cell.

[0192] According to embodiments of this application, the gene-edited organism includes transgenic animals or transgenic plants and their derivative products, such as organs, tissues / cells obtained therefrom, or seeds produced therefrom.

[0193] According to embodiments of this application, based on edited cells, this method can obtain transgenic organisms, including transgenic animals and transgenic plants, thereby having broader application prospects in agriculture, biomedical research and other fields.

[0194] To make it easier to understand, the process of constructing transgenic animals or plants is briefly described below:

[0195] According to embodiments of this application, the transgenic process can use genetically transformable plant or animal materials as recipients, including animal fertilized eggs or embryos, and plant embryogenic tissues, vegetative bodies, protoplasts, mature leaves, etc. According to embodiments of this application, the animals that can be used include, but are not limited to, pigs, non-human primates, dairy cows, sheep, goats, rabbits, etc., and the plants that can be used include Arabidopsis thaliana, tobacco, tomato, rice, wheat, corn, soybeans, cotton, bananas, etc.

[0196] According to embodiments of this application, gene editing-related components can be efficiently and accurately introduced through methods such as microinjection, viral vector delivery, electroporation, and Agrobacterium-mediated transformation to perform gene editing on the recipient.

[0197] Next, by cultivating gene-edited cells, transgenic animal individuals or plant plants can be obtained. The gene-edited individuals can then be screened to obtain organisms with specific genetic improvements. Specifically, methods such as PCR, DNA sequencing, protein immunoblotting, and mass spectrometry can be used to verify and screen the edited animals and plants, ensuring the accuracy and effectiveness of gene editing.

[0198] According to the embodiments of this application, the resulting non-human animals, plants or their offspring can have broad application potential in fields such as animal model preparation, drug production, and agricultural production.

[0199] Gene-edited cells

[0200] In a seventh aspect of this application, a gene-edited cell is disclosed. According to embodiments of this application, the cell is obtained by the methods described in the second and third aspects of this application, or by gene editing of cells using the composition described in the first aspect of this application.

[0201] According to embodiments of this application, gene-edited cells effectively avoid adverse consequences caused by DNA double-strand cutting, such as adverse events caused by DNA double-strand breaks, such as additional chromosomal translocations or inversions. At the same time, they also effectively avoid the problem of low gene insertion efficiency caused by single-strand breaks (SSBs) being quickly repaired back to the unedited state, making them difficult to apply alone to gene editing.

[0202] According to embodiments of this application, compared to traditional methods that rely on DNA double-strand breaks, the viability, proliferation rate, and total number of cells edited using this method are also improved.

[0203] According to embodiments of this application, no adverse events caused by DNA double-strand cutting, such as additional chromosomal translocations or inversions, occur in the cells.

[0204] According to embodiments of this application, the cell is a non-reproductive cell.

[0205] According to embodiments of this application, the given site is a cell engineering editing site, a safe harbor site, or a gene site related to a patient's disease.

[0206] According to embodiments of this application, the cells include patient-derived or allogeneic stem cells, induced pluripotent stem cells (iPSCs), cancer cells, and immune cells.

[0207] Gene-edited cell population

[0208] In an eighth aspect of this application, a gene-edited transgenic cell population is provided. According to embodiments of this application, the gene-edited cell population is obtained by the methods described in the second and third aspects of this application, or by gene editing of the cell population using the composition described in the first aspect of this application.

[0209] According to embodiments of this application, gene-edited cell populations effectively avoid adverse consequences caused by DNA double-strand cutting, such as adverse events caused by DNA double-strand breaks, such as additional chromosomal translocations or inversions. At the same time, they also effectively avoid the problem of low gene insertion efficiency caused by single-strand breaks (SSBs) being quickly repaired back to the unedited state, making them difficult to apply alone to gene editing.

[0210] According to embodiments of this application, no adverse events caused by DNA double-strand cutting, such as additional chromosomal translocations or inversions, occur in the cell population.

[0211] According to embodiments of this application, compared to traditional methods that rely on DNA double-strand breaks, the viability, proliferation rate, and total number of cells edited using this method are also improved.

[0212] According to embodiments of this application, the cell population is a non-reproductive cell population, including organoids and tissues.

[0213] According to embodiments of this application, the given site is a cell engineering editing site, a safe harbor site, or a gene site related to a patient's disease.

[0214] According to embodiments of this application, the cell population includes patient-derived or allogeneic stem cell populations, induced pluripotent stem cell (iPSC) populations, cancer cell populations, and immune cell populations.

[0215] Genetically modified organisms

[0216] In a ninth aspect of this application, a genetically modified organism (GMO) is proposed. According to an embodiment of this application, the GMO is constructed using the method of the sixth aspect.

[0217] According to embodiments of this application, the gene-edited organism includes transgenic animals or transgenic plants and their derivative products, such as organs, tissues / cells obtained therefrom, or seeds produced therefrom.

[0218] According to embodiments of this application, based on edited cells, this method can obtain transgenic organisms, including transgenic animals and transgenic plants, thereby having broader application prospects in agriculture, biomedical research and other fields.

[0219] Methods for constructing recombinant immune cells and the recombinant immune cells obtained

[0220] In another aspect of this application, a method for constructing recombinant immune cells in vivo or in vitro is provided, comprising:

[0221] Cells are treated with a gene-editing composition to perform targeted gene editing at at least one given site on the cells to obtain edited recombinant immune cells. The gene-editing composition comprises: a nicking enzyme complex or its encoding nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule that can be linked to or bound to a second recruitment component, the first and second recruitment components being adapted to form a specific binding, wherein the nicking enzyme complex is adapted to create a single-stranded nick in the genome, without simultaneously cleaving double-stranded DNA, the nicking enzyme complex being configured to be linked to the first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be linked to or bound to the second recruitment component, the first and second recruitment components being adapted to form a specific binding.

[0222] According to embodiments of this application, the cells include at least one of the following: T cells, NK cells, DNT cells, γδT cells, iPSC cells, stem cells, iPSC-derived immune cells, umbilical cord blood-derived T cells, NK T cells (T cells expressing NK cell surface markers), virus-specific memory T cells, CD8-positive and CD4-positive naive T cells (TN), central memory T cells (TCM), Stem-like memory T cells (TSCM), DC cells, microphages, TIL cells, CIK cells, autologous immune cells, allogeneic immune cells, universal immune cells, CAR-T cells, STAR-T cells, TCR-T cells, universal CAR-T cells, CAR-M cells, and CAR-NK cells.

[0223] According to embodiments of this application, the recombinant immune cells are CAR-T, universal CAR-T, CAR-NK, STAR-T, CAR-M, or TCR-T.

[0224] According to embodiments of this application, the method includes the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3 alpha (DNMT3A), Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-b receptor II, protein tyrosine phosphatase 1B (PTP1B), CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein(PRDM1), CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL 2. FAS, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-β receptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP At least one of the following gene editing methods is performed: P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1, and TIGER. This involves single-site or multi-site gene editing.

[0225] According to embodiments of this application, the recombinant immune cells are adapted to overexpress or knock down immunomodulatory factors or genes that regulate the functional activity of immune cells.

[0226] According to embodiments of this application, the active regulatory factors and / or immunomodulatory factors are selected from CD47, ADR, NR4A family, HLA-E, B2M-HLA-E, NK inhibitory ligand, IL-2, IL-12, IL-15, IL-18, IL21, type 1 cytokines, type 2 cytokines, engineered IL2 superkine Super2, IL33, IL25, Flt3L, IFNγ, IL-7 / CCL19, IFNα, IFNβ, TNF, NKG2A, CD16, mbIL-15, iCasp9, RQR8, HSV-TK (inserted transgenes), TGFβ-Trap (such as dnTGFβRII or TGFβR-KRAB), IL-23, IL-9, GM-CSF, CCL17, CCL22, XCL1, PD-1 dominant negative receptor, CTLA-4 dominant negative receptor. receptor (CTLA-4 DNR), TIM-3 DNR, LAG-3 DNR, anti-PD-1 antibody fragment (scFv), anti-CTLA-4 antibody fragment, OX40L (CD252), 4-1BBL (CD137L), CD80, CD86, ICOS, ICOSL, CD40L, EGFRt, CD20t, SynNotch, BiKE, Flagellin, suicide gene, tetracycline-induced expression system (Tet-On / Tet-Off), miR-155 sponge, c-JUN Overexpression, PD-L1, CXCL9, CXCL10, CXCL11, CD137(4-1BB) antigen receptor, HVEM (TNFRSF14), BTLA, VISTA, CD73, CD39, IDO1, Arginase-1, FASL, Galectin-9, TIM-1, TIM-4, SHP-1, SHP-2, SOCS1, SOCS3, T-bet, Eomes, STAT3, HIF-1α, FOXP3, Nef, BNLF2a, US2, US11, K5, NKG2D-DAP10 signaling complex, CD94 / NKG2C and regulatory miRNA, and at least one of viral-derived signaling or regulatory proteins.

[0227] According to embodiments of this application, the TGFβ-Trap includes dnTGFβRII or TGFβR-KRAB.

[0228] According to embodiments of this application, the regulatory miRNA includes at least one of miR-146a and miR-21.

[0229] According to embodiments of this application, the viral signaling or regulatory protein is selected from the Nef protein derived from HIV-1 Clade B, the BNLF2a protein derived from EBV, the US2 and US11 proteins derived from hCMV, and the K5 protein derived from KSV.

[0230] According to embodiments of this application, the method includes the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein (CISH), DNA methyltransferase 3 alpha (DNMT3A), Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-β receptor II, protein tyrosine phosphatase 1B (PTP1B), CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein(PRDM1), CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA(CD25), IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2 , FAS, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP At least one of the following gene editing methods is performed: P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1, and TIGER. This involves single-site or multi-site gene editing.

[0231] According to embodiments of this application, the gene editing includes: inserting a CAR / TCR / STAR gene that targets and recognizes the edited cell and / or inserting a gene that regulates the function of immune cells in killing target cells and / or a gene that regulates the functional activity of immune cells.

[0232] According to embodiments of this application, the genes include BCMA-CAR, CD20-CAR, CD22-CAR, CD30-CAR, CD123-CAR, CD38-CAR, CD7-CAR, ROR1-CAR, CD70-CAR, CD19-CAR, ERBB2-CAR, CD3-CAR, mesothelin-CAR, CD7-CAR, CD138-CAR, MICA-CAR, ULBP1-CAR, ULBP6-CAR, CD33-CAR, CD123-CAR, FLT3-CAR, BCMA-CAR, CD5-CAR, NKG2D CAR, CS1-CAR, 1XX-CAR, BiTE, TCR, STAR, and TCR. mimic antibody gene, optimized TCRα / β chain gene, HaloTag-CAR, SNAP-TAG-CAR, synotch, CLEC12A(CLL-1)-CAR, HER2-CAR, MSLN-CAR, PSMA-CAR, CEA-CAR, MUC1-CAR, IL13Ra2-CAR, CD171(L1CAM)-CAR, NKG2D-CAR, GD2-CAR, EGFR / EGFRvIII-CAR, CD33-CAR, CD44v6-CAR, FLT3-CAR, CD117-CAR, CD200-CAR, CD1a-CAR, Siglec-6-CAR, B7-H3-CAR, CLDN18.2-CAR, FOLR1-CAR, MAGE-A4-CAR, EPCAM-CAR, PD-L1-CAR, B7-H4-CAR, CAIX-CAR, CD133-CAR, uPAR-CAR, Tandem-CAR, Dual-CAR, Split-CAR, SUPRA-CAR, UniCAR, SpyTag-CAR, FITC-CAR, ON-switch CAR, OFF-switch CAR, iCasp9-CAR, TRUCK-CAR, Armored-CAR, γδT-CAR, MAIT-CAR, iNKT-CAR, IL1RAP-CAR, CSF1R-CAR, CD276-CAR, PTK7-CAR, STEAP1-CAR, ROR2-CAR, TEM8-CAR, AXL-CAR, ANXA2-CAR, PRAME-CAR, TAG72-CAR, TSPAN8-CAR, TM4S F1-CAR, BSP-CAR, NTRK1-CAR, GD3-CAR, LMP1-CAR, NY-ESO-1-CAR, c-Met-CAR, VEGFR2-CAR, CDH17-CAR, ICAM-1-CAR, switchable-CAR, separable-CAR, inhibitory-CAR(iCAR), CAR-anti-PD1, masked-CAR, light-activated CAR, hypoxia-responsive At least one of CAR, FAP-CAR, Nef-CAR, and GPC3-CAR.

[0233] According to embodiments of this application, the genes that can regulate the function of immune cells in killing target cells and / or regulate the functional activity of immune cells include at least one of the following: co-stimulatory molecules, transcription factors, active regulatory factors, immunomodulatory factors, and virus-derived signaling or regulatory proteins.

[0234] According to embodiments of this application, the co-stimulatory molecule is selected from at least one of OX40, ICOS, and CD27.

[0235] According to embodiments of this application, the transcription factor is selected from at least one of BATF, TFAP4, and FOXO1.

[0236] According to embodiments of this application, the active regulatory factors and / or immunomodulatory factors are selected from CD47, ADR, NR4A family, HLA-E, B2M-HLA-E, NK inhibitory ligand, IL-2, IL-12, IL-15, IL-18, IL21, type 1 cytokines, type 2 cytokines, engineered IL2 superkine Super2, IL33, IL25, Flt3L, IFNγ, IL-7 / CCL19, IFNα, IFNβ, TNF, NKG2A, CD16, mbIL-15, iCasp9, RQR8, HSV-TK, TGFβ-Trap, IL-23, IL-9, GM-CSF, CCL17, CCL22, XCL1, PD-1 dominant negative receptor, CTLA-4 dominant negative receptor, TIM-3 DNR, LAG-3 DNR, anti-PD-1 antibody fragment, anti-CTLA-4 antibody fragment, OX40L, 4-1BBL, CD80, CD86, ICOS, ICOSL, CD40L, EGFRt, CD20t, SynNotch, BiKE, Flagellin, suicide gene, tetracycline-induced expression system, miR-155 sponge, c-JUN Overexpression, PD-L1, CXCL9, CXCL10, CXCL11, CD137 antigen receptor, HVEM, BTLA, VISTA, CD73, CD39, IDO1, Arginase-1, FASL, Galectin-9, TIM-1, TIM-4, SHP-1, SHP-2, SOCS1, SOCS3, T-bet, Eomes, STAT3, HIF-1α, FOXP3, Nef, BNLF2a, US2, US11, K5, NKG2D-DAP10 signaling complex, CD94 / NKG2C, and at least one of the following:

[0237] According to embodiments of this application, the viral regulatory or signaling protein is selected from the Nef protein derived from HIV-1 Clade B, the BNLF2a protein derived from EBV, the US2 and US11 proteins derived from hCMV, and the K5 protein derived from KSV.

[0238] According to embodiments of this application, the recombinant immune cells obtained by the above method can effectively avoid the adverse consequences caused by DNA double-strand cutting, such as additional chromosomal translocations and inversions. It also effectively promotes homologous recombination / homologous repair based on single-strand breaks, solving the problem of low gene insertion efficiency caused by the rapid repair of single-strand breaks (SSBs) back to an unedited state, making them difficult to apply alone to gene editing. Furthermore, the inventors of this application unexpectedly discovered that the recombinant immune cells obtained using the above method exhibit higher survival rates and cell viability. This is of great significance for the engineering of immune cells, especially the preparation of CAR-T cells, as it can improve cell yield and quality, thereby enhancing the efficiency and safety of the entire cell therapy.

[0239] Therefore, this application proposes a method for constructing recombinant immune cells in vitro, which is particularly suitable for site-specific gene editing of immune cells to enhance their anti-tumor capabilities. This method involves using a special gene-editing composition capable of producing single-strand cuts in DNA instead of double-strand breaks, thereby reducing genomic instability and other adverse consequences. According to embodiments of this application, the above-described method for constructing recombinant immune cells has at least one of the following advantages:

[0240] 1. The precision and safety of gene editing

[0241] According to embodiments of this application, the method of the present invention can reduce insertions or deletions: high-throughput sequencing shows that CRISPR enIE-edited cells have fewer insertions or deletions, demonstrating higher editing accuracy.

[0242] According to embodiments of this application, the method of the present invention can reduce the activation of the cytoplasmic cGAS-STING pathway: compared with Cas9, the expression of IFNB1 in CRISPR enIE-edited cells is significantly reduced, indicating that the cGAS-STING pathway is less activated.

[0243] According to embodiments of this application, the method of the present invention can reduce apoptosis: the expression of the apoptosis indicator NOXA is also lower, indicating that CRISPR enIE editing leads to less apoptosis.

[0244] 2. Cell viability and survival rate

[0245] According to embodiments of this application, the method of the present invention can improve cell viability: the viability of recombinant T cells obtained by the CRISPR enIE method is 4.3 times that of the Cas9 method, indicating that the CRISPR enIE method is significantly superior to the Cas9 method in maintaining cell viability.

[0246] According to embodiments of this application, the method of the present invention can improve cell viability: 8 days after transfection, the total number of cells treated with CRISPR enIE was 18.2 times that of cells treated with Cas9, demonstrating the advantage of the CRISPR enIE method in maintaining cell viability.

[0247] 3. Proliferative capacity

[0248] According to the embodiments of this application, the method of the present invention can enhance proliferation capacity: CRISPR enIE-edited T cells began to show a significant trend of increased proliferation capacity on the 4th day after electroporation, and on the 8th day, the total number of cells increased significantly, showing strong proliferation capacity.

[0249] Therefore, according to the embodiments of this application, the method of the present invention can improve the efficiency and safety of cell therapy, and increase cell yield and quality. This is particularly important for the preparation of CAR-T cells, as it can improve cell yield and quality, thereby improving treatment efficiency and safety: by improving cell survival rate and viability, it further improves the overall efficiency and safety of cell therapy. According to the embodiments of this application, since the CRISPR enIE method improves cell viability and proliferation efficiency, this method is very suitable for industrialization needs, supporting large-scale production of recombinant immune cells. Higher cell viability and proliferation efficiency mean that more target cells can be obtained in a shorter time, thereby improving production efficiency. It can also reduce cell culture and processing time and reduce cell loss, helping to reduce overall production costs. In addition, the high precision and low cell damage characteristics of the CRISPR enIE method help improve the quality and consistency of the final product.

[0250] Cell therapy involves giving autologous or allogeneic cells new abilities to treat a variety of diseases. This includes stem cell therapy, where stem cells are used for tissue repair and regeneration due to their potential for self-renewal and multi-lineage differentiation. Cell therapy can also include immune cell therapy, such as chimeric antigen receptor T-cell (CAR-T) therapy, which uses a patient's own T cells that have been genetically engineered to enhance their ability to recognize and attack cancer cells.

[0251] Non-viral, programmable large-fragment DNA insertion can improve cell-based therapies such as CAR-T, CAR-NK, STAR-T, and TCR-T cell engineering. It can enhance the stability and function of engineered immune cells while reducing the risk of random insertion. However, current large-fragment insertion methods primarily rely on DNA double-strand cutting techniques, which increase the risks of genomic instability, toxicity, and translocation. The gene-editing method proposed in this application avoids double-strand cutting, providing a safer option for T-cell engineering.

[0252] Therefore, another aspect of this application proposes recombinant immune cells and recombinant immune cell populations that can be obtained by the above methods. These recombinant immune cells and cell populations have the following characteristics and potential applications:

[0253] According to embodiments of this application, these recombinant immune cells include, but are not limited to, T cells, natural killer cells (NK cells), CD3+CD4-CD8- double-negative T cells (DNT cells), T cells performing innate immune functions (γδT cells), iPSC cells, stem cells, iPSC-derived immune cells, umbilical cord blood-derived T cells, NK T cells (T cells expressing NK cell surface markers), virus-specific memory T cells, CD8-positive and CD4-positive naive T cells (TN cells), central memory T cells (TCM), stem-like memory T cells (TSCM), dendritic cells (DC cells), microphages, tumor-infiltrating lymphocytes (TIL cells), cytokine-induced killer cells (CIK cells), chimeric antigen receptor T cells (CAR-T cells), CAR-M, and chimeric antigen receptor natural killer cells (CAR-NK cells). According to embodiments of this application, these recombinant immune cells have enhanced anti-tumor capabilities through site-specific gene editing, for example, by inserting CAR genes or other enhancing genes.

[0254] According to embodiments of this application, recombinant immune cell populations can be customized to meet different therapeutic needs, such as CAR-T cell populations targeting specific tumor antigens. According to embodiments of this application, because the CRISPR enIE method improves cell viability and proliferation efficiency, the resulting recombinant immune cell populations meet the needs of clinical applications in both quantity and quality.

[0255] According to embodiments of this application, these recombinant immune cells and cell populations can be used to develop novel cancer immunotherapies, such as CAR-T cell therapy, which directly targets tumor cells. They can also enhance the function of NK cells or T cells through gene editing for the treatment of viral or bacterial infections. By regulating the function of immune cells, they may have potential value in the treatment of certain autoimmune diseases.

[0256] As mentioned earlier, the CRISPR enIE method is adaptable to large-scale production, providing sufficient immune cells and cell populations for clinical applications. Increased production efficiency and reduced manufacturing costs make these treatments more cost-effective, potentially improving patient accessibility.

[0257] For ease of understanding, the following is a detailed description of an example of a method for constructing recombinant immune cells using the gene editing system of this application: The process of constructing universal CAR-T cells can be described in detail below, using the gene editing system of this invention as an example:

[0258] First, methods such as electroporation, liposome transfection, lipid nanoparticle transfection, viral transfection, and microinjection can be used to effectively introduce the encoding nick enzyme complex and target nucleic acid molecules (such as CAR genes) into immune cells.

[0259] According to embodiments of the present invention, the construction of universal CAR-T cells involves the use of the following genes: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3 alpha (DNMT3A), Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-β receptor II, protein tyrosine phosphatase 1B (PTP1B), CD2, TCRab / CD3, CD5, CS1, IL-6, and PR domain finger. protein(PRDM1), CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP Editing of single or multiple sites of genes including P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1, and TIGER. The selection of these sites aims to reduce the immune rejection response of allogeneic CAR-T cells while maximizing therapeutic efficacy. Next, at the selected gene sites, the CAR gene is inserted using gene editing technology, enabling T cells to recognize and target specific cancer cells. This step is crucial for CAR-T cell therapy, allowing T cells to recognize and kill cancer cells in a highly specific manner. According to embodiments of the present invention, this step can simultaneously inactivate endogenous genes during gene insertion, such as inactivating endogenous T cell receptors while inserting CAR at the TRAC site.

[0260] Subsequently, flow cytometry or PCR technology was used to screen and identify the edited immune cells to ensure that the CAR gene had been successfully inserted and expressed. These technologies can rapidly and accurately assess the efficiency and specificity of gene editing.

[0261] Then, the proliferation capacity of CAR-T cells was assessed by measuring changes in the number of CAR-T cells under antigen stimulation through cell counting. Next, CAR-T cells were co-cultured with target cells in vitro, and the release of lactate dehydrogenase in the culture supernatant was measured to evaluate the CAR-T cell killing ability against target cells. Cytokine secretion concentrations were detected using ELISA (enzyme-linked immunosorbent assay), and changes in CAR-T cell activation and depletion phenotypes were detected by flow cytometry to determine the intensity and persistence of CAR-T cell tumor-killing ability. This step was used to assess the responsiveness of CAR-T cells to specific antigens, ensuring their activity and function in vivo.

[0262] Ultimately, the edited cells can be used to prepare tumor therapeutic agents or to establish immune cell libraries, providing resources for future research and treatment. These applications demonstrate the broad potential of edited immune cells.

[0263] use

[0264] This application discloses the use of cells constructed by the methods described herein, or the cells described herein, in the preparation of pharmaceuticals for treating diseases, including genetic abnormalities such as gene mutation-related diseases, cancer, and autoimmune diseases. According to embodiments of this application, targeted repair of a patient's cells can be effectively achieved, thereby improving the efficiency of disease treatment.

[0265] According to embodiments of this application, the disease is a single-gene disease or a polygenic disease.

[0266] According to embodiments of this application, the diseases include cancer, hematologic disorders, autoimmune diseases, deafness, eye diseases, metabolic diseases, AIDS, and musculoskeletal disorders. Optionally, they include sickle cell anemia and thalassemia, hereditary retinal diseases, Duchenne muscular dystrophy, amyotrophic lateral sclerosis (ALS), myotonic dystrophy type 1 / II, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type 1, lymphoma, B-cell leukemia and multiple myeloma, bladder cancer, liver cancer, lung cancer, epithelial cancer, colon cancer, cervical cancer, severe combined immunodeficiency, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type 1, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, systemic vasculitis, and type 1 diabetes.

[0267] Example

[0268] Example 1: Improving the insertion efficiency of large DNA fragments using the CRISPR enIE system

[0269] Referring to Figure 7, this embodiment aims to verify the effectiveness of the CRISPR-based enIE (enhanced nickase-mediated insertion editing) system in improving the efficiency of large DNA fragment insertion mediated by single-strand nicks.

[0270] Example 1.1:

[0271] The TALE protein was used as the first recruitment component, and the DNA sequence that TALE specifically binds to was used as the second recruitment component. Referring to Figure 7a, TALE was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with TALE-specific binding sequences.

[0272] Experimental steps:

[0273] Constructing nCas9(D10A)-TALE and gRNA expression plasmids: Design plasmids, add a linker (50-200 bp) between nCas9(D10A) and TALE, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0274] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid includes a TALE-specific binding sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0275] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0276] Transfection of cells: The donor DNA template and nCas9-TALE-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0277] Screening and identification: Puromycin was used for screening after transfection.

[0278] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0279] Example 1.2:

[0280] ZF protein was used as the first recruitment component, and the DNA sequence that ZF specifically binds to was used as the second recruitment component. Referring to Figure 7a, ZF was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with ZF-specific binding sequences.

[0281] Experimental steps:

[0282] Constructing nCas9(D10A)-ZF and gRNA expression plasmids: Design plasmids to add a linker sequence (50-200bp) between nCas9(D10A) and ZF, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0283] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid includes a ZF-specific binding sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0284] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0285] Transfection of cells: The donor DNA template and nCas9-ZF-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0286] Screening and identification: Puromycin was used for screening after transfection.

[0287] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0288] Example 1.3:

[0289] ParB protein was used as the first recruitment component, and the ParS sequence as the second recruitment component. Referring to Figure 7a, ParB was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with ParS.

[0290] Experimental steps:

[0291] Constructing nCas9(D10A)-ParB and gRNA expression plasmids: Design plasmids to add a linker sequence (50-200 bp) between nCas9(D10A) and ParB, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0292] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid contains 8 repeats of the ParS sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0293] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0294] Transfection of cells: The donor DNA template and nCas9-ParB-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0295] Screening and identification: Puromycin was used for screening after transfection.

[0296] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0297] Example 1.4:

[0298] LacI protein was used as the first recruitment component, and the lacO sequence was used as the second recruitment component. Referring to Figure 7a, LacI was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with lacO.

[0299] Experimental steps:

[0300] Constructing nCas9(D10A)-LacI and gRNA expression plasmids: Design plasmids to add a linker (50-200 bp) between nCas9(D10A) and LacI, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0301] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid includes the lacO sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0302] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0303] Transfection of cells: The donor DNA template and nCas9-LacI-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0304] Screening and identification: Puromycin was used for screening after transfection.

[0305] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0306] Example 1.5:

[0307] Monomeric streptavidin was used as the first recruitment component, and biotin as the second recruitment component. Referring to Figure 7a, monomeric streptavidin mSA was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit biotin-modified double-stranded DNA donors (dsDNA donors).

[0308] Experimental steps:

[0309] Constructing nCas9(D10A)-mSA and gRNA expression plasmids: Design plasmids to add a linker (50-200 bp) between nCas9(D10A) and mSA, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0310] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence, which includes an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0311] Preparation of donor DNA: Synthesize modified PCR primers and produce modified donor DNA by PCR amplification.

[0312] Transfection of cells: The donor DNA template and nCas9-mSA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0313] Screening and identification: Puromycin was used for screening after transfection.

[0314] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0315] Example 1.6:

[0316] The HIV Gag protein was used as the first recruitment component, and the Ψ(Psi) sequence was used as the second recruitment component. Referring to Figure 7a, the Gag protein was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with the Psi sequence.

[0317] Experimental steps:

[0318] Constructing nCas9(D10A)-Gag protein and gRNA expression plasmids: Design plasmids to add a linker (50-200 bp) between nCas9(D10A) and Gag protein, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0319] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid includes the Ψ (Psi) sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0320] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0321] Transfection of cells: The donor DNA template and nCas9-Gag protein-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0322] Screening and identification: Puromycin was used for screening after transfection.

[0323] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0324] Example 1.7:

[0325] The large T antigen of SV40 was used as the first recruitment component, and the SV40 Ori sequence was used as the second recruitment component. Referring to Figure 7a, the large T antigen was fused with SpCas9 nickase (Sp nCas9 D10A) to recruit double-stranded DNA donors (dsDNA donors) with the SV40 Ori sequence.

[0326] Experimental steps:

[0327] Constructing nCas9(D10A)-Gag protein and gRNA expression plasmids: Design plasmids to add a linker (50-200 bp) between nCas9(D10A) and the large T antigen, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0328] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid includes the SV40 Ori sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0329] Preparation of donor DNA: Synthesize PCR primers and amplify the donor DNA by PCR.

[0330] Transfection of cells: The donor DNA template and nCas9-large T antigen-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0331] Screening and identification: Puromycin was used for screening after transfection.

[0332] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0333] Example 1.8:

[0334] MCP was used as the first recruitment component, and MS2 as the second recruitment component. Referring to Figure 7a, MCP was fused with SpCas9 nickase (Sp nCas9D10A) to recruit RNA donors with the MS2 sequence.

[0335] Experimental steps:

[0336] Constructing nCas9(D10A)-MCP protein and gRNA expression plasmids: Design plasmids to add a linker (50-200 bp) between nCas9(D10A) and MCP, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0337] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid contains six repeats of the MS2 sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0338] Preparation of donor RNA: An in vitro transcription template for donor RNA is prepared by PCR amplification, and donor RNA with MS2 sequence, homologous arms at both ends and target sequence is produced by in vitro transcription.

[0339] Transfection of cells: The donor RNA template and nCas9-MCP-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0340] Screening and identification: Puromycin was used for screening after transfection.

[0341] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0342] Example 1.9:

[0343] PCP was used as the first recruitment component, and PP7 as the second recruitment component. Referring to Figure 7a, PCP was fused with SpCas9 nickase (Sp nCas9D10A) to recruit RNA donors with the PP7 sequence.

[0344] Experimental steps:

[0345] Constructing nCas9(D10A)-PCP protein and gRNA expression plasmids: Design plasmids to add a linker sequence (50-200bp) between nCas9(D10A) and PCP, and include nuclear localization sequences (NLS) to promote gene editing in the cell nucleus.

[0346] Preparation of donor template plasmid: Construct a plasmid containing the donor template sequence. This plasmid contains six repeats of the PP7 sequence, an enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene or other target sequence, and homologous arms of 100-1500 bp on each side. Design the homologous arms so that the target insertion site is within 20 bp of the nick site.

[0347] Preparation of donor RNA: An in vitro transcription template for donor RNA is prepared by PCR amplification, and donor RNA with PP7 sequence, homologous arms at both ends and target sequence is produced by in vitro transcription.

[0348] Transfection of cells: The donor RNA template and nCas9-PCP-gRNA plasmid were transfected into the cells using liposome transfection reagent. Transfection was performed when the cells reached 85% confluence.

[0349] Screening and identification: Puromycin was used for screening after transfection.

[0350] Verify editing effect: Analyze the proportion of cells expressing EGFP by flow cytometry or by sequencing analysis to assess gene insertion efficiency.

[0351] Experimental Results: Exemplary results from this embodiment are shown in Figure 7. The results demonstrate that the gene editing system of this application can insert the target gene at an endogenous site within the cell. After inserting the EGFP gene at the endogenous gene site, flow cytometry analysis revealed that the editing efficiency of the CRISPR enIE system is approximately 5.2 times that of using nCas9 alone. This embodiment demonstrates that the enIE system can significantly improve the efficiency of homology repair at single nick sites, enabling efficient insertion of large DNA fragments via homology repair. By optimizing the interaction between the nick enzyme and donor DNA in the CRISPR-Cas9 system, the enIE system significantly improves the efficiency of large DNA fragment insertion, providing a new strategy for the development of gene editing technology.

[0352] Example 2: CRISPR enIE can efficiently insert large DNA fragments and minimize the risk of insertion or deletion mutations.

[0353] This embodiment was performed using essentially the same method as in Example 1, with large-fragment insertion. The results are shown in Figure 8. Sequencing results indicate that the CRISPR enIE system can minimize the risk of insertion or deletion while achieving high insertion efficiency. In target sites where no target gene was inserted, Cas9 resulted in a high proportion (99.7%) of insertion or deletion mutations, while CRISPR enIE resulted in only 0.46% of insertion or deletion mutations, comparable to the mutation rate of a single nCas9 (0.37%). This proportion is only 1 / 217 of that of Cas9 (99.7%) and 1 / 49.8 of that of paired nCas9 (22.9%), as detailed in Figure 8a.

[0354] Experimental steps:

[0355] This embodiment was carried out using essentially the same method as in Example 1. After inserting large fragments of DNA, cells were collected for genome extraction.

[0356] Cell collection: Discard the culture medium, rinse with PBS, add 0.25% trypsin for digestion, stop digestion with culture medium after 1 minute, centrifuge and discard the culture medium.

[0357] Genomic DNA extraction: Genomic DNA was extracted using commercially available genomic DNA extraction kits.

[0358] Nested PCR construction of sequencing libraries: First, amplicons without the target sequence and those containing the target sequence are generated using primers designed on the outer homologous arms. The amplicons are then subjected to agarose gel electrophoresis, and the amplicons without the target sequence are recovered by gel excision. Next, using the recovered amplicons as PCR templates, a second round of PCR is performed: upstream primers containing the P5 adaptor and i5 index and downstream primers containing the P7 adaptor and i7 index are designed at 70-100 bp on both sides of the cut to generate amplicons of approximately 200-400 bp. These amplicons constitute the sequencing library.

[0359] Secondary sequencing: The sequencing library was sent to the company for next-generation sequencing using the Illumina PE150 strategy.

[0360] CRISPResso2: Input the results of next-generation sequencing into this website for data analysis.

[0361] Referring to Figure 8b, further sequencing revealed that precisely inserted products dominated the CRISPR enIE edited products, with the ratio of targeted edited products to non-targeted edited products being 20.2 times higher than that of products edited with Cas9. In contrast, non-targeted edited products predominated after Cas9 editing. These results demonstrate that the CRISPR enIE system efficiently and safely performs precise insertion at a single nick site.

[0362] Example 3: CRISPR enIE efficiently and with low risk inserts large DNA fragments into different sites, and can mediate the efficient and low-risk insertion of DNA fragments >10kb.

[0363] This embodiment was carried out using essentially the same method as in Example 1, and large-fragment DNA insertion was performed.

[0364] This embodiment further tested the insertion capability at other sites. The DNA donor and plasmid preparation methods, as well as the transfection method, were the same as in Example 1. The experimental results further demonstrate that the CRISPR enIE system has the ability to efficiently and with low risk integrate genes at different sites.

[0365] Furthermore, CRISPR enIE has been shown to effectively mediate the insertion of larger DNA fragments. In this example, when inserting a 10.2kb fragment, the enIE group also achieved a higher insertion efficiency than Cas9. Moreover, while effectively inserting large fragments, the mutation risk of CRISPR enIE is significantly lower than that of Cas9.

[0366] Example 4: Repeated Editing

[0367] In this embodiment, the enIE system was used for repeated editing to improve editing efficiency. After Cas9 editing, most sites where no gene was inserted mutated, thus disrupting the gRNA recognition sequence. The results after enIE editing were completely different; almost all sites where no gene was inserted retained the wild type. Therefore, enIE makes it possible to improve overall editing efficiency by re-editing after the initial gene editing (see Figure 6). In this embodiment, a second editing was performed 14 days after the first gene editing at a specific site, and the experimental procedure was the same as in Example 1. The efficiency of the second CRISPR enIE editing was improved by 1.7 times (see Figure 9). This result demonstrates that enIE can repeatedly edit unedited sites, thereby improving overall editing efficiency.

[0368] Example 5: The improved version enIE2 further improves the efficiency of large gene fragment insertion.

[0369] Methods such as using tandem nucleic acid aptamers, tandem protein tags, polyproteins, or phase-separating proteins can be employed to enable nCas9(D10A) to recruit more DNA donors.

[0370] Referring to Figure 5, based on Example 1, the number of DNA donors linked to the second recruitment component recruited by the nicking enzyme complex through the specific interaction between the first recruitment component and the second recruitment component is increased.

[0371] Two to 50 tandem nucleic acid aptamers are added to the 3' end of the sgRNA, and a first recruitment component expressing the binding protein of nCas9(D10A) and the fusion nucleic acid aptamer is expressed. Binding sequences of the first recruitment component are added to both ends of the donor DNA. nCas9 forms a complex with the sgRNA. Each nucleic acid aptamer can bind up to two binding proteins of the nucleic acid aptamer. A single sgRNA recruits up to 100 nucleic acid aptamer-binding protein combinations through specific binding of the nucleic acid aptamer and protein. Then, up to 60 DNA donors are recruited through the interaction of the first recruitment component and the second recruitment component. It is also possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1. This application provides an exemplary demonstration, as shown in Examples 5.1 to 5.8.

[0372] A first recruitment component with 2-50 tandem repeats is fused to the C-terminus of nCas9(D10A), and a second recruitment component with 1-50 tandem repeats is added to both ends of the donor DNA. This allows a single nCas9-tandem first recruitment component to bind to 2-100 DNA donors with the second recruitment component, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1. This application provides an illustrative example, as shown in Examples 5.9-5.26.

[0373] Example 5.1:

[0374] Eight tandem MS2 sequences were added to the 3' end of the sgRNA, and nCas9 (D10A) and the TALE protein fused with MCP were expressed, respectively. TALE protein binding sequences were added to both ends of the donor DNA. nCas9 and sgRNA formed a complex, and each MS2 sequence could bind up to two MCP proteins. A single sgRNA recruited up to 16 MCP-TALE proteins through the specific binding of MS2-MCP, and then recruited multiple DNA donors through the interaction of TALE protein-TALE specific binding sequences. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0375] Example 5.2:

[0376] Eight tandem PP7 sequences were added to the 3' end of the sgRNA, and nCas9 (D10A) and TALE proteins fused with PCP were expressed, respectively. TALE protein binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, and each PP7 sequence could bind up to two PCP proteins. A single sgRNA recruited up to 16 PCP-TALE proteins through the specific binding of PP7-PCP, and then recruited multiple DNA donors through the interaction of TALE protein-TALE specific binding sequences. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0377] Example 5.3:

[0378] Eight tandem MS2 sequences were added to the 3' end of the sgRNA, and nCas9(D10A) and the ZF protein fused with MCP were expressed, respectively. ZF protein binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, and each MCP sequence could bind up to two MS2 proteins. A single sgRNA recruited up to 16 MCP-ZF proteins through the specific binding of MS2-MCP, and then recruited multiple DNA donors through the interaction of ZF protein-ZF specific binding sequences. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0379] Example 5.4:

[0380] Eight tandem PP7 sequences were added to the 3' end of the sgRNA, and nCas9 (D10A) and ZF proteins fused with PCP were expressed, respectively. ZF protein binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, and each PP7 sequence could bind up to two PCP proteins. A single sgRNA recruited up to 16 PCP-ZF proteins through the specific binding of PP7-PCP, and then recruited multiple DNA donors through the interaction of ZF protein-ZF specific binding sequences. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0381] Example 5.5:

[0382] Eight tandem MS2 sequences were added to the 3' end of the sgRNA, and nCas9(D10A) and LacI proteins fused with MCP were expressed, respectively. Sixteen tandem repeats of lacO binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, with each MS2 sequence binding to up to two MCP proteins. A single sgRNA recruited up to 16 MCP-LacI proteins through the specific binding of MS2-MCP, and subsequently recruited multiple DNA donors through LacI-lacO interactions. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0383] Example 5.6:

[0384] Eight tandem PP7 sequences were added to the 3' end of the sgRNA, and nCas9(D10A) and PCP-fused LacI proteins were expressed, respectively. Sixteen tandem repeats of lacO binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, with each PP7 sequence binding to up to two PCP proteins. A single sgRNA recruited up to 16 PCP-LacI proteins through the specific binding of PP7-PCP, and subsequently recruited multiple DNA donors through LacI-lacO interactions. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0385] Example 5.7:

[0386] Eight tandem MS2 sequences were added to the 3' end of the sgRNA, and nCas9(D10A) and TetR proteins fused with MCP were expressed, respectively. Sixteen tandem repeats of tetO binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA formed a complex, with each MS2 sequence binding to up to two MCP proteins. A single sgRNA recruited up to 16 MCP-TetR proteins through the specific binding of MS2-MCP, and subsequently recruited multiple DNA donors through TetR-tetO interactions. It is also possible that the DNA donors act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site.

[0387] Example 5.8:

[0388] Eight tandem PP7 sequences were added to the 3' end of the sgRNA, and nCas9(D10A) and the TetR protein fused with PCP were expressed, respectively. Sixteen tandem repeats of tetO binding sequences were added to both ends of the donor DNA. The nCas9 and sgRNA form a complex, with each PP7 sequence binding to up to two PCP proteins. A single sgRNA recruits up to 16 PCP-TetR proteins through the specific binding of PP7-PCP, and then recruits multiple DNA donors through TetR-tetO interactions. It may also amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade of DNA donor-bridge interactions.

[0389] Example 5.9:

[0390] A ParB protein with three tandem repeats was fused to the C-terminus of nCas9(D10A), and 16 tandem repeats of the parS sequence were added to both ends of the donor DNA. This allows a single nCas9-tandem ParB protein to bind to multiple DNA donors with the ParS sequence, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1.

[0391] Example 5.10:

[0392] Three tandem repeats of LacI protein were fused to the C-terminus of nCas9(D10A), with 16 tandem repeats of lacO sequence added to both ends of the donor DNA. This allows a single nCas9-tandem LacI protein to bind to multiple DNA donors with lacO sequences, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1.

[0393] Example 5.11:

[0394] A TetR protein with three tandem repeats was fused to the C-terminus of nCas9(D10A), and 16 tandem repeats of the tetO sequence were added to both ends of the donor DNA. This allows a single nCas9-tandem TetR protein to bind to multiple DNA donors with tetO sequences, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1.

[0395] Example 5.12:

[0396] Three tandem repeats of mSA were fused to the C-terminus of nCas9(D10A). A single nCas9-tandem mSA can bind multiple biotin-modified DNA donors and may recruit multiple nicks-donor DNA complexes at the target site through a cascade amplification via DNA donors as bridges. Other steps are the same as in Example 1.

[0397] Example 5.13:

[0398] Three tandem repeats of TALE protein were fused to the C-terminus of nCas9(D10A), with TALE-specific binding sequences added to both ends of the donor DNA. This allows a single nCas9-tandem TALE protein to bind to multiple DNA donors with TALE-specific binding sequences, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1.

[0399] Example 5.14:

[0400] Three tandem repeats of ZF protein were fused to the C-terminus of nCas9(D10A), with ZF-specific binding sequences added to both ends of the donor DNA. This allows a single nCas9-tandem ZF protein to bind to multiple DNA donors with ZF-specific binding sequences, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade amplification via the DNA donor as a bridge. Other steps are the same as in Example 1.

[0401] Example 5.15:

[0402] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with ParB protein was expressed. Sixteen tandem repeats of the parS sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-ParB, recruiting DNA donors with the parS sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0403] Example 5.16:

[0404] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with LacI protein was expressed. Sixteen tandem repeats of the lacO sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-LacI molecules, recruiting DNA donors with the lacO sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0405] Example 5.17:

[0406] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with the TetR protein was expressed. Sixteen tandem repeats of the tetO sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-TetRs, recruiting DNA donors with the tetO sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0407] Example 5.18:

[0408] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a GCN4-targeting scFV fused with mSA protein was expressed. The donor DNA was biotin-modified. A single nCas9-tandem GCN4 can bind multiple scFV-mSA, recruiting biotin-modified DNA donors, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade of DNA donors acting as bridges. Other steps were the same as in Example 1.

[0409] Example 5.19:

[0410] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with the TALE protein was expressed. TALE-specific binding sequences were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-TALEs, recruiting DNA donors with TALE-specific binding sequences. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0411] Example 5.20:

[0412] Eight tandem repeats of the GCN4 peptide were fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with ZF protein was expressed. ZF-specific binding sequences were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-ZF, recruiting DNA donors with ZF-specific binding sequences. It is possible that the DNA donor can act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0413] Example 5.21:

[0414] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with ParB protein was expressed. Sixteen tandem repeats of the parS sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-ParB, recruiting DNA donors with the parS sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0415] Example 5.22:

[0416] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with LacI protein was expressed. Sixteen tandem repeats of the lacO sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-LacI molecules, recruiting DNA donors with the lacO sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0417] Example 5.23:

[0418] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with TetR protein was expressed. Sixteen tandem repeats of the tetO sequence were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-TetRs, recruiting DNA donors with the tetO sequence. It is possible that the DNA donor acts as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0419] Example 5.24:

[0420] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a GCN4-targeting scFV fused with mSA protein was expressed. The donor DNA was biotin-modified. A single nCas9-tandem GCN4 can bind multiple scFV-mSA, recruiting biotin-modified DNA donors, and may amplify the recruitment of multiple nickase-donor DNA complexes at the target site through a cascade of DNA donors acting as bridges. Other steps are the same as in Example 1.

[0421] Example 5.25:

[0422] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with TALE protein was expressed. TALE-specific binding sequences were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-TALEs, recruiting DNA donors with TALE-specific binding sequences. It is possible that the DNA donor can act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0423] Example 5.26:

[0424] A GCN4 peptide with 24 tandem repeats was fused to the C-terminus of nCas9(D10A), and a scFV targeting GCN4 fused with ZF protein was expressed. ZF-specific binding sequences were added to both ends of the donor DNA. A single nCas9-tandem GCN4 can bind multiple scFV-ZF, recruiting DNA donors with ZF-specific binding sequences. It is possible that the DNA donor can act as a bridge to amplify the recruitment of multiple nickase-donor DNA complexes at the target site. Other steps are the same as in Example 1.

[0425] Experimental results: enIE2 significantly improved the insertion efficiency of large DNA fragments. For example, the insertion efficiency of the EGFP gene was 1.5 times that of CRISPR enIE and 3.76 times that of Cas9 (see Figure 10). This indicates that by recruiting more donors at the nick site, enIE2 can further influence the selection of DNA repair pathways, thereby leading to higher gene insertion efficiency.

[0426] Example 6: Application of CRISPR enIE in the treatment of genetic diseases: Integrating therapeutic gene fragments into safe sites and restoring normal gene expression in a cell model simulating the disease.

[0427] Integrating disease-treating DNA into specific locations in the genome can correct many diseases caused by gene mutations. CRISPR enIE can be used to treat stable and sustained gene expression, providing long-term or even permanent solutions for diseases that previously required continuous treatment.

[0428] This embodiment uses the same DNA donor, plasmid preparation, and transfection methods as in Example 1 to test the efficiency of inserting the HBB gene into a safe site. Mutations or deletions of the HBB gene are associated with sickle cell disease and beta-thalassemia, and inserting normal HBB into a safe site is a promising treatment for these diseases. This embodiment then validates the system in a cell model simulating lysosomal acid lipase deficiency (LAL-D). Mutations in the LIPA gene can cause this disease. This embodiment uses CRISPR enIE to insert wild-type LIPA gene cDNA into a safe site. Referring to Figure 11, RT-qPCR detection shows that CRISPR enIE-mediated gene editing can completely restore wild-type LIPA mRNA expression, with an efficiency 3.3 times that of Cas9. Single-cell sorting and PCR identification show that the LIPA cDNA cell insertion efficiency is 6.2 times that of Cas9, proving that the wild-type LIPA cDNA was inserted into a safe site. This embodiment also tested the effect of inserting wild-type LIPA cDNA at the natural LIPA site. After CRISPR enIE editing, wild-type LIPA mRNA was partially restored in the LIPA-mutant cell line. Furthermore, this embodiment also verified the effectiveness of this strategy in mouse cells, achieving gene insertion at a safe site in mice.

[0429] Example 7: Application of CRISPR enIE in the field of immune cell engineering: It increased the number of cells with large fragment insertions in human primary T cells with low toxicity.

[0430] Non-viral, programmable large-fragment DNA insertion can improve cell-based therapies such as CAR-T and TCR-T cell engineering. It can enhance the stability and function of engineered immune cells while reducing the risk of random insertion. However, current large-fragment DNA insertion methods primarily rely on DNA double-strand cutting techniques, which increase the risk of genomic instability, toxicity, and translocation. The CRISPR enIE method avoids induced double-strand cutting, providing a safer option for T-cell engineering. This embodiment applies this strategy to primary T cells.

[0431] Experimental steps:

[0432] The method is basically the same as in Example 1, and the preparation method of the DNA donor is the same as in Example 1. However, in this example, the enIE system is delivered to T cells by in vitro assembly of RNP or by using mRNA.

[0433] In vitro expression of protein components: In this example, the enIE protein was expressed and purified in Escherichia coli.

[0434] In vitro transcription of gRNA and assembly of transfection system: gRNA is prepared using an in vitro transcription kit and then mixed with DNA donor, or gRNA prepared by in vitro transcription is mixed with mRNA prepared by in vitro transcription and DNA donor.

[0435] Electrotransfection: The assembled system was transfected into 3 x 10 cells using an electrotransfection system.

[0436] CRISPR enIE successfully edited the EGFP gene. T cells edited by CRISPR enIE exhibited significantly better cell viability than those edited by Cas9, showing a 4.3-fold increase (see Figure 12a); the number of cells in the CRISPR enIE group was 7.3 times that of the Cas9 group (see Figure 12b). Furthermore, the number of EGFP-positive cells produced by the CRISPR enIE group was 2.8 times that of the Cas9 group (see Figure 12c). These results demonstrate that CRISPR enIE not only avoids the detrimental effects of Cas9 system-edited T cells but also ensures successful editing to produce a greater number of viable T cells, making it a promising tool for cell-based therapy.

[0437] Example 8: Multi-segment insertion

[0438] This embodiment further expands the application strategy of enIE, enabling simultaneous editing of multiple different sites in a single transfection, and further improves its editing effect through secondary editing and recruiting more repair templates by nCas9.

[0439] Inserting genes at multiple loci can advance the treatment of complex genetic diseases or optimize immunotherapies for cancer, such as generating universal CAR-T cells. However, using DNA double-strand cutting-based methods to simultaneously insert two fragments can lead to increased translocations and potential oncogenic risks. This embodiment utilizes the enIE system based on nickase to systematically mitigate these unintended editing outcomes. Therefore, this embodiment investigates its potential to simultaneously insert large sequences into two different genomic sites.

[0440] This embodiment was carried out using essentially the same method as in Example 1, but the plasmid used simultaneously expressed gRNA targeting two sites and was simultaneously transfected with donor DNA targeting two sites.

[0441] After enIE editing, reporter genes were successfully inserted into 6.5% of cells at gene loci on two different chromosomes, Chr7 and Chr15. Building on this, enIE2 further improved the effectiveness of multi-site editing. The number of cells simultaneously edited at Chr7 and Chr15 increased to 2.1 times that of enIE (see Figure 13a).

[0442] This embodiment further compares the translocation risk of the Cas9 and CRISPR enIE systems during two-site gene insertion. This embodiment found that Cas9 editing produces translocation products that can be detected by PCR, while CRISPR enIE editing produces undetectable translocation products (see Figure 13b). This indicates that CRISPR enIE is safer than Cas9 when performing simultaneous multi-site editing.

[0443] Example 9:

[0444] Example 9 is the same as Example 1, except that the length of the inserted fragment is 100 bp. The other steps are basically the same as in the example. This example demonstrates that the enIE system can significantly improve the efficiency of gene insertion at single nick sites and can efficiently insert large DNA fragments through homologous sequence-guided repair.

[0445] Example 10:

[0446] Example 10 is the same as Example 1, except that the length of the inserted fragment is 200 bp. The other steps are basically the same as in the example. This example demonstrates that the enIE system can significantly improve the efficiency of gene insertion at single nick sites and can efficiently insert large DNA fragments through homologous sequence-guided repair.

[0447] Example 11:

[0448] Example 11 is the same as Example 1, except that the length of the inserted fragment is 300 bp. The other steps are basically the same as in the example. This example demonstrates that the enIE system can significantly improve the efficiency of gene insertion at single nick sites and can efficiently insert large DNA fragments through homologous sequence-guided repair.

[0449] Example 12:

[0450] Example 12 is the same as Example 1, except that the length of the inserted fragment is 500 bp. The other steps are basically the same as in the example. This example demonstrates that the enIE system can significantly improve the efficiency of gene insertion at single nick sites and can efficiently insert large DNA fragments through homologous sequence-guided repair.

[0451] Example 13:

[0452] Example 13 is the same as Example 1, except that the length of the inserted fragment is 1kb. The other steps are basically the same as in the example. This example demonstrates that the enIE system can significantly improve the efficiency of gene insertion at single nick sites and can efficiently insert large DNA fragments through homologous sequence-guided repair.

[0453] Example 14: This example is the same as Example 7, except that human primary T cells are replaced with NK cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA fragment insertion is increased with low toxicity in NK cells.

[0454] Example 15: This example is the same as Example 7, except that human primary T cells are replaced with DC cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA fragment insertion is increased with low toxicity in DC cells.

[0455] Example 16: This example is the same as Example 7, except that human primary T cells are replaced with TIL cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA fragment insertion is increased with low toxicity in TIL cells.

[0456] Example 17: This example is the same as Example 7, except that human primary T cells are replaced with CIK cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA fragment insertion is increased with low toxicity in CIK cells.

[0457] Example 18:

[0458] Example 18.1: This example is the same as Example 7, except that human primary T cells are replaced with CAR-T cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA fragment insertion is increased with low toxicity in CAR-T cells.

[0459] Example 18.2: This example is the same as Example 7, except that human primary T cells are replaced with CAR-NK cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA insertion is increased with low toxicity in CAR-NK cells.

[0460] Example 19: This example is the same as Example 7, except that human primary T cells are replaced with hematopoietic stem cells. The other processes are the same as in Example 7. The results show that the number of cells with large DNA insertion is increased with low toxicity in hematopoietic stem cells.

[0461] Example 20: Evaluating the efficiency of the CRISPR enIE system and the Cas9 system at therapeutic gene insertion safety sites.

[0462] In this embodiment, the efficiency of inserting therapeutic genes into safe sites using the gene editing system of this application, following the methods described above, is demonstrated. Mutations in the IL2RG gene are associated with X-linked severe combined immunodeficiency (X-SCID), and inserting normal IL2RG cDNA into safe sites is a possible therapeutic approach. In this embodiment, a DNA donor expressing IL2RG cDNA was used, and the IL2RG cDNA was inserted into safe sites using the same plasmid preparation and transfection methods as in Example 1. The edited cells were isolated into 96-well plates by flow cytometry and cultured into monoclonal cell lines, followed by PCR to identify the insertion efficiency. Schematic results are shown in Figure 14, showing that editing using CRISPR enIE is 2.9 times more efficient than Cas9 editing. This embodiment demonstrates that the CRISPR enIE system can efficiently insert therapeutic genes into safe sites and also illustrates that the CRISPR enIE system exhibits significantly higher editing efficiency than the Cas9 system. This indicates that the enIE system has higher precision and efficiency in gene editing, which may be attributed to the higher specificity and accuracy of the enIE system in recognizing and cutting target DNA sequences.

[0463] Example 21: Assessing gene expression levels after inserting the therapeutic gene into a safe site.

[0464] In this embodiment, based on Example 14, the expression level of the gene after inserting the therapeutic gene into a safe site is further evaluated. In short, this embodiment includes culturing gene-edited cells under standard conditions, extracting total RNA from the edited cells, synthesizing cDNA using the extracted RNA, and then quantifying the expression level of the IL2RG gene using RT-qPCR technology. The illustrative results are shown in Figure 15, which displays the relative expression levels of the IL2RG gene in the control group (Ctrl), the Cas9 editing group, and the enIE editing group. The IL2RG gene expression level in the enIE editing group was significantly higher than that in the Cas9 editing group and the control group. The expression level in the enIE editing group was 2.7 times that of the Cas9 editing group and was significantly different from that in the control group.

[0465] As a result, cells edited using the CRISPR enIE system showed higher IL2RG gene expression levels, indicating that the system can effectively promote the expression of therapeutic genes. This high expression level may help improve or cure diseases caused by IL2RG gene mutations, such as X-SCID. This further confirms the superiority of the CRISPR enIE system in enhancing gene expression.

[0466] Example 22: Evaluation of the efficiency of inserting the therapeutic gene (LIPA cDNA) into a safe site in a cell line carrying a pathogenic mutation using the gene editing system of this application.

[0467] In this embodiment, the efficiency of inserting the therapeutic gene (LIPA cDNA) into a safe site in a cell line carrying a pathogenic mutation gene is assessed.

[0468] Materials and Methods:

[0469] DNA donor preparation: Constructing DNA donors expressing normal LIPA cDNA.

[0470] Plasmid preparation: Plasmids were prepared using the same method as in Example 1.

[0471] Cell transfection: The LIPA cDNA was inserted into a safe site in the target cells using the same transfection method.

[0472] Cell isolation and culture: Edited cells were isolated by flow cytometry and cultured in 96-well plates to form monoclonal cell lines.

[0473] Insertion efficiency determination: The insertion efficiency of LIPA cDNA was determined using PCR technology.

[0474] The illustrative results are shown in Figure 16, which show that the editing efficiency of the CRISPR enIE editing system is 6.2 times that of the Cas9 system.

[0475] Thus, the CRISPR enIE system showed significantly higher editing efficiency than the Cas9 system, indicating that the enIE system has higher precision and efficiency in gene editing.

[0476] Example 23: Evaluation of expression levels when normal LIPA cDNA is inserted into the natural site of the gene using the gene editing system of this application.

[0477] In this embodiment, based on Example 22, the expression level of normal LIPA cDNA inserted into the natural site of the gene using the gene editing system of this application is further evaluated.

[0478] In this embodiment, RT-qPCR was used to quantify the LIPA gene expression level. The results are shown in Figure 17, which displays the relative LIPA gene expression levels in WT (wild-type cells), Mut (cells carrying the pathogenic mutant gene), Ctrl (non-targeted cells carrying the pathogenic mutant gene), and the enIE editing group. The LIPA gene expression level in the enIE editing group was significantly higher than that in the Mut and Ctrl groups, but lower than that in the WT group. The expression level in the enIE editing group was several times that of the Ctrl group, and this difference was statistically significant compared to the WT group. The results demonstrate that cells edited using the CRISPR enIE system can partially restore normal gene expression.

[0479] Example 24: Evaluation of the effectiveness of CRISPR enIE for programmable gene knock-in in primary T lymphocytes

[0480] The implementation method of this embodiment is basically the same as that of embodiment 7. The difference is that the inventors evaluated the effect of CRISPR enIE in primary T lymphocytes for programmable gene knock-in in this embodiment.

[0481] This application employs conventional methods to determine cell viability and obtains the corresponding experimental results through flow cytometry analysis. It should be noted that cells with EGFP fragment insertion exhibit fluorescence, allowing for direct acquisition of efficiency. Cell viability is determined by staining cells with a Fixable Viability Stain 510, followed by flow cytometry analysis. First, the total cell count is obtained using a cell counter; then, the live cell number is calculated by multiplying the viability by the total cell number. The EGFP value is calculated by multiplying the positive cell rate by the total cell number. + Cell number. The T cell growth curve was obtained by counting cells using a cytometer. Schematic results are shown in Figure 18, illustrating the editing efficiency and cell viability in T cells using Cas9 and the CRISPR enIE of this invention. EGFP was successfully inserted into T cells using CRISPR enIE, with a cell viability 4.3 times higher than that of cells edited with Cas9, and the total number of EGFP-positive cells was 2.1 times higher. This shows that although the insertion efficiency of CRISPR enIE is slightly lower than that of Cas9, its edited cells have higher activity, ultimately resulting in a higher yield of edited cells.

[0482] The results showed that the CRISPR enIE method significantly improved cell viability by reducing the risk of genomic instability, toxicity, and chromosomal translocations by avoiding induced DNA double-strand breaks (DSBs). This result suggests that the CRISPR enIE method may be a safer and more effective approach to T-cell engineering, particularly in applications requiring the maintenance of cell viability and function, such as CAR-T and TCR-T cell therapies. The high cell viability and safety of the CRISPR enIE method make it an attractive option for cell therapies like CAR-T and TCR-T cell therapies, where maintaining cell viability and function is crucial.

[0483] Example 25: Evaluation of the proliferative capacity of CRISPR enIE in T cells

[0484] The method used is basically the same as in Example 1. The preparation method of the DNA donor is the same as in Example 1. The difference is that in this example, the enIE system is delivered to T cells by in vitro assembly of RNP or by using mRNA during cell transfection.

[0485] In vitro expression of protein components: In this example, the enIE protein was expressed and purified in Escherichia coli.

[0486] In vitro transcription of gRNA and assembly of transfection system: gRNA is prepared using an in vitro transcription kit and then mixed with DNA donor, or gRNA prepared by in vitro transcription is mixed with mRNA prepared by in vitro transcription and DNA donor.

[0487] Electroporation: The assembled system was transfected into 3 x 10⁶ cells using an electroporation system. Cell counts were performed on days 2, 4, 6, and 8 post-electropy using a cell counter. The fold change in T cell count for each treatment method was calculated and statistically analyzed.

[0488] The results showed that CRISPR enIE-edited T cells began to exhibit a significant proliferation trend on day 4 after electroporation, and by day 8, the total number of cells was 10.2 times that of Cas9-treated cells. The CRISPR enIE method demonstrated significantly superior proliferation capacity in T cells compared to the Cas9 method, as shown in Figure 19. This result indicates that the CRISPR enIE method not only performs better in maintaining cell viability but also exhibits a significant advantage in long-term proliferation capacity. Therefore, the CRISPR enIE method may be more suitable for cell therapy applications requiring large-scale proliferation, such as CAR-T and TCR-T cell therapies, which is of great significance for developing more effective immunocellular therapy strategies. By using the CRISPR enIE method, more target cells can be obtained in a shorter time, thereby improving the efficiency and effectiveness of treatment and meeting the needs of industrialization.

[0489] Example 26: Evaluation of the safety of CRISPR enIE in gene editing

[0490] The method used is basically the same as in Example 1. The preparation method of the DNA donor is the same as in Example 1. However, in this example, the enIE system is delivered to T cells by in vitro assembly of RNP or the use of mRNA and electroporation.

[0491] In vitro expression of protein components: In this example, the enIE protein was expressed and purified in Escherichia coli.

[0492] In vitro transcription of gRNA and assembly of transfection system: gRNA is prepared using an in vitro transcription kit and then mixed with DNA donor, or gRNA prepared by in vitro transcription is mixed with mRNA prepared by in vitro transcription and DNA donor.

[0493] Electrotransfection: The assembled system was transfected into 3 x 10 cells using an electrotransfection system.

[0494] Three days after electroporation, edited T cells were harvested and their genomes extracted. Library construction was performed using PCR, followed by high-throughput sequencing to assess insertions and deletions. Twenty-four hours after electroporation, cells were harvested and mRNA extracted. The expression levels of IFNB1 and NOXA were analyzed using RT-qPCR or RNA sequencing. Differences in insertion / deletion, IFNB1, and NOXA expression in T cells treated by each method were calculated and statistically analyzed. The results are shown in Figure 20. CRISPR enIE-edited cells showed fewer non-target insertions and deletions, indicating higher editing precision. The significantly reduced IFNB1 expression in CRISPR enIE-edited cells indicated lower activation of the cGAS-STING pathway. Lower NOXA expression in CRISPR enIE-edited cells also suggested less apoptosis.

[0495] The results showed that CRISPR enIE-edited cells had fewer insertions or deletions. Furthermore, compared to Cas9, CRISPR enIE-edited cells exhibited significantly reduced expression of IFNB1, a marker protein of the cGAS-STING pathway, indicating lower activation of this pathway. Additionally, the expression of the apoptosis indicator NOXA was also lower, suggesting that CRISPR enIE editing induced less apoptosis. These results demonstrate that CRISPR enIE has higher safety for gene editing.

[0496] Therefore, the CRISPR enIE method exhibits higher safety in gene editing, mainly in the following aspects: fewer insertions or deletions, improving editing precision; significantly reduced IFNB1 expression, indicating lower activation of the cGAS-STING pathway; and lower NOXA expression, indicating a lower incidence of apoptosis. These results suggest that the CRISPR enIE method may cause less cell damage during gene editing and is more suitable for applications requiring the preservation of cell viability and function, such as immunotherapy.

[0497] Example 27: Preparation of CAR-T cells using CRISPR enIE

[0498] Editing was performed using CRISPR enIE and Cas9. The steps in this example were basically the same as in Example 7, the difference being that the DNA donor used was an anti-CD19 CAR gene. Immunostaining was performed on the CAR gene on the surface of T cells, and flow cytometry was used to analyze the editing efficiency. The efficiency of CAR insertion by CRISPR enIE was slightly lower than that by Cas9, but there was no statistically significant difference. Furthermore, the cell viability of CRISPR enIE-edited cells was 3.1 times that of Cas9-edited cells, and both the number of viable cells and the total number of CAR-T cells were significantly increased, as shown in Figure 21. This demonstrates that CRISPR enIE editing has advantages over Cas9 in terms of cell viability and yield when preparing CAR-T cells.

[0499] Example 28: Preparation of CAR-T cells using CRISPR enIE and CRISPR enIE2

[0500] To further improve the efficiency of CAR-T cell preparation, the steps in this embodiment are basically the same as in Example 27. This embodiment optimizes the delivery method; the optimized CRISPR enIE and CRISPR enIE2 further improve the efficiency by 2.0 times and 3.6 times, respectively, as shown in Figure 22, enabling efficient CAR-T cell preparation. Furthermore, CRISPR enIE and enIE2 also further increase the total yield of CAR-T cells.

[0501] Example 29: Detection of the killing ability of CAR-T cells prepared by CRISPR enIE

[0502] The steps in this embodiment are basically the same as in Example 27. In this embodiment, CAR-T cells were co-cultured with the CD-19-expressing human B lymphoma cell line Raji for 48 hours at different effector-to-target ratios (E:T ratios) of 1:3, 1:2, 1:1, 2:1, and 3:1. Compared with CAR-T cells (LV-19BBz-CAR-T) prepared using the traditional lentiviral method, CAR-T cells (enIE-19BBz-CAR-T) edited by CRISPR enIE showed improved tumor cell killing ability, especially at low effector-to-target ratios, as shown in Figure 23. This demonstrates that CAR-T cells prepared by CRISPR enIE possess tumor-killing ability and show promise as an alternative to the traditional lentiviral method.

[0503] Example 30: Construction of a universal CAR-T using CRISPR enIE in combination with base editing

[0504] Using CRISPR enIE in conjunction with base editing, gene knockout was achieved simultaneously with CAR insertion via CRISPR enIE and base editing at sites such as TRAC, B2M, and PDCD1. The steps in this embodiment are essentially the same as in Example 27, the difference being the addition of a base editor to the CRISPR enIE. Immunostaining of T cell surfaces and analysis of editing efficiency using flow cytometry and sequencing showed that effective base editing and knockout could be achieved simultaneously with CAR insertion.

[0505] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," 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.

[0506] 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 composition for gene editing of cells, characterized in that, include: A nicking enzyme complex or its encoded nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule containing a target site homologous sequence, the target nucleic acid molecule being connectable to or bound to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding. The nicking enzyme complex is adapted to generate single-stranded DNA nicks in the genome, without simultaneously cleaving double-stranded DNA, and is configured to be linked to a first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be linked to or bind to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

2. The composition according to claim 1, characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: Nucleic acid fragments and proteins that specifically recognize said nucleic acid fragments; Protein tag - the ligand, nucleic acid aptamer, or binding protein of the protein tag; Antigen-antibody; Optional modified non-natural amino acids-corresponding binding ligands; Optional modified non-natural bases-corresponding binding ligands; Sequence-specific DNA / RNA binding proteins and their specific binding sequences; Structure-specific DNA / RNA binding proteins and their specific binding sequences; Specific nucleic acid modifications and corresponding binding proteins, antibodies, or ligands of these modifications; Viral proteins or their functional fragments interact with corresponding cellular or soluble viral receptors; Viral proteins and their specific antibodies; Viral envelope proteins and target cell membrane lipid / protein components; Viral proteins - DNA / RNA sequences recognized by viral proteins; Small molecules and protein or nucleic acid sequences that specifically recognize the molecule; Optionally, the cells are cells carrying pathogenic genes in the body. Optionally, the gene editing is used to repair the pathogenic gene; Optionally, the pathogenic gene is a single-gene disease pathogenic gene or a polygenic disease pathogenic gene; Optionally, the gene editing is performed in vitro, within cells, or in vivo.

3. The composition according to claim 1 or 2, characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: The Rep protein-PCV recognition sequence from porcine circovirus 2PCV; TALE and its specific binding sequence; ZF and its specific binding sequence; CRISPR proteins and variants and their specific recognition sequences; ParB-parS; LacI-lacO; TetR-tetO; CymR-cuO; Non-natural amino acids containing azido groups and ligands containing alkyne groups; non-natural amino acids containing azido groups and non-natural bases containing alkyne groups; non-natural amino acids containing cyclopropene groups and tetrazine compounds; non-natural amino acids containing trans-cyclooctene and S-tetrazine; non-natural amino acids containing azido groups and cyclopropane-cyclooctene; non-natural amino acids containing tetrazine groups and cyclopropene or trans-cyclooctene. AeF-DBCO; AzF-DBCO; NAEK-DBCO; HaloTag - Chlorinated alkane ligand; SnapTag - Corresponding ligand; TMP-Tag-corresponding ligand; Avidin and biotin; Monomeric or non-monomeric or tandem streptavidin-biotin; Viral DNA / RNA binding proteins - their binding sequences or DNA / RNA components; Viral or cellular-derived DBP, SV40, NS1, ICP8, ORF, E1, E2, integrate, UL9, HMG, Rep, NS1, IE175, NP, N, NS, GAG, RRM, HLH, AT-hook motif, EBNA1 protein - its protein recognition sequence or component. SunTag-anti-GCN4 single-chain variable sequence antibody; MoonTag-gp41 nanobody.

4. The composition according to any one of claims 1 to 3, characterized in that, The first recruitment component is covalently linked to the nick enzyme.

5. The composition according to claim 4, characterized in that, The first recruitment component and the nick enzyme constitute a fusion protein. Optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; Optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; Optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; Optionally, the first recruitment component includes at least one Rep protein, and the second recruitment component is a Rep protein recognition sequence; Optionally, the first recruitment component includes at least one monomeric or non-monomeric streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; Optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; Optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; Optionally, the first recruitment component may include a combination of multiple individual units connected in series.

6. The composition according to claim 5, characterized in that, The first recruitment component and the nick enzyme are connected by a linker.

7. The composition according to claim 6, characterized in that, The linker has 15-150 amino acids.

8. The composition according to claim 7, characterized in that, The linker has the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence that is at least 50% homologous to SEQ ID NO: 1, for example, at least 80% homologous, for example, at least 90% homologous, or at least 95% homologous.

9. The composition according to any one of claims 1 to 3, characterized in that, The first recruitment component is non-covalently linked to the nick enzyme. Optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; Optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; Optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; Optionally, the first recruitment component includes at least one viral protein, and the second recruitment component is a viral protein recognition sequence; Optionally, the first recruitment component is a monomeric or non-monomer streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; Optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; Optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ sequence; Optionally, the first recruitment component may include a combination of multiple individual units connected in series.

10. The composition according to any one of claims 1 to 9, characterized in that, The directed nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases; Optionally, the first recruitment component is directly or indirectly linked to the binding proteins of the plurality of said nucleic acid aptamers or to modifications or ligands of non-natural bases; Optionally, the directed nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases, such as at least one, at least two, or 3 to 100 of the nucleic acid aptamers or non-natural bases.

11. The composition according to claim 10, characterized in that, The combination of nucleic acid aptamers and binding proteins includes at least one of the following: MS2-MCP, PP7-PCP, BoxB-lambdaN, Com-com, Streptavidin aptamer-streptavidin.

12. The composition according to claim 11, characterized in that, The binding protein of the nucleic acid aptamer binds to the protein tag, the first recruitment component is linked to at least one protein tag binding fragment, and the protein tag binding protein is adapted to specifically bind to the protein tag.

13. The composition according to any one of claims 1-12, characterized in that, The nicking enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment; Optionally, the first recruitment component may be directly or indirectly linked to a plurality of proteins that specifically recognize the protein tag.

14. The composition according to claim 13, characterized in that, The protein tag binding fragment includes at least one of the following types that specifically recognize the protein tag: scFv, Fab, Fab', F(ab')2, Fv, Nanobody, bispecific antibody, Minibodies, single-domain antibody, the protein tag binding protein; Optionally, the combination of proteins that specifically recognize the protein tag includes at least one of the following: GCN4 - recognizes scFVs, nanobodies, or their respective variants of GCN4; gp41 - recognizes scFVs, nanobodies, or their respective variants of gp41.

15. The composition according to any one of claims 9, 13-14, characterized in that, The protein tag is at least one of the GCN4 sequence and the gp41 sequence.

16. The composition according to claim 15, characterized in that, The nick enzyme is fused with at least one GCN4 sequence and / or gp41 sequence, and the first recruitment component is fused with an scFv or a variant thereof that specifically recognizes the GCN4 sequence and / or gp41 sequence.

17. The composition according to claim 13, characterized in that, The nicking enzyme is fused with a plurality of the protein tags, for example, at least one, at least two, or 2 to 30 of the protein tags.

18. The composition according to any one of claims 1 to 3, characterized in that, The first recruitment component and the second recruitment component constitute a fusion protein.

19. The composition according to any one of claims 1 to 18, characterized in that, The nicking enzyme includes at least one of the following: a CRISPR-based nicking enzyme variant, a TALE or ZF nicking enzyme that cleaves single-stranded DNA, a homing endonuclease variant that cleaves only one strand of DNA, MagaTAL, ARCUS, or a complex containing the above nicking enzyme variants, as well as a combination of the above nicking enzyme variants and complexes with a homologous recombination repair factor. in, Optionally, the CRISPR-based nicking enzymes include SpCas9(D10A), SpCas9(H840A), and nicking enzyme variants of SaCas9, SpG Cas9, SpRY Cas9, ScCas9, Cas12, Cas13, CasX, CasMINI, IscB, and TnpB. Optionally, the CRISPR protein, TALE, or ZF cleavage enzyme that cuts single-stranded DNA includes CRISPR, TALE, and ZF variants based on MutH, FokI, I-AniI, I-OnuI, and I-TevI. Optionally, the nick enzyme has the protein / amino acid sequence shown in SEQ ID NO: 2; Optionally, the system has multi-core positioning signals; Optionally, the nicking enzyme comprises a combination of the above-mentioned nicking enzyme variants and homology repair-promoting protein factors, including Rad51, Rad51(A190L A192L), MRN Complex(MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51(S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 proteins or variants thereof; Optionally, the target nucleic acid molecule is plasmid DNA, viral DNA / RNA, double-stranded DNA, or single-stranded DNA / RNA; Optionally, the target nucleic acid molecule contains a homologous sequence of the target site, which can be used as a template for homologous recombination / homologous repair / gene editing, and provides a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a specified site through homologous repair or for performing base substitution, repair or small fragment insertion, replacement or repair on a specific site through homologous repair. Optionally, the target nucleic acid molecule contains a homologous sequence of the target site, which can be used as a template for homologous recombination / homologous repair / gene editing, and provides a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a specified site through homologous recombination or for inserting, replacing or repairing a large fragment at a specific site through homologous recombination. Optionally, the exogenous nucleic acid sequence has homologous arms at both ends, and the homologous arms are homologous to the nucleic acid sequence of a specific region in the target genome. When the target nucleic acid molecule is used to insert a large fragment with a gene size greater than 500 bp, the length of the homologous arm is 100-1500 bp. When the target nucleic acid molecule is used for base substitution, repair or small fragment insertion, the homologous arm can also be 15-1000 bp. Optionally, the insertion site of the exogenous nucleic acid sequence is within 100 bp of the DNA single-strand nick site; Optionally, the target nucleic acid molecule carries a identifiable modification or a specific protein-binding sequence; Optionally, the target nucleic acid molecule is prepared by plasmid or PCR.

20. The composition according to any one of claims 1-19, characterized in that, include: A delivery system that loads the nick enzyme complex or its encoded nucleic acid molecule and the target nucleic acid molecule.

21. The composition according to any one of claims 1-19, characterized in that, The composition is suitable for use in conjunction with base editing, lead editing, and RNA editing regulation technologies.

22. The composition according to claim 20, characterized in that, The nucleic acid molecule encoding the nick enzyme complex is located on DNA or RNA. Optionally, the nucleic acid molecule encoding the nick enzyme complex is plasmid DNA; Optionally, the nucleic acid molecule encoding the nick enzyme complex is a viral vector DNA / RNA; Optionally, the nucleic acid molecule encoding the nick enzyme complex is RNA; Optionally, the nick enzyme complex is a protein or a protein-RNA complex.

23. The composition according to claim 20, characterized in that, The delivery system is suitable for liposome transfection, electrotransfection, cationic polymer transfection, microinjection, lipid nanoparticle transfection, and viral vector delivery.

24. The composition according to claim 22, characterized in that, The mass ratio of the plasmid to the target nucleic acid molecule is (1-10):(1-10); Optionally, the mass ratio of the RNA to the target nucleic acid molecule is (1-10):(1-10); Optionally, the mass ratio of the protein or protein-RNA complex to the target nucleic acid molecule is (1-10):(1-10).

25. A method for gene editing in cells, characterized in that, include: Contact the composition of any one of claims 1 to 24 with a cell to introduce a nick enzyme complex and a target nucleic acid molecule into the cell for gene editing at at least one given site in the cell.

26. The method according to claim 25, characterized in that, The given site includes: a safe harbor site, a cell engineering editing site, and a disease gene site. Optionally, the contact is performed in vitro or in vivo. Optionally, the cells are derived from a patient or allogeneic organism, and the cells are introduced into the patient after contact with the composition according to any one of claims 1 to 24.

27. The method according to claim 25, characterized in that, The given site includes at least one of the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3alpha, Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerolkinases, adenosine A2A receptor ID3, SOX4, TGF-β receptor II, protein tyrosine phosphatase 1B, CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein, CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OTUD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, APP, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1 and TIGER.

28. The method according to claim 25, characterized in that, The gene editing includes introducing point mutations, gene insertions, deletions, or substitutions at at least one of the given sites.

29. The method according to claim 25, characterized in that, The gene editing includes inserting a nucleic acid fragment of not less than 100 bp, for example, not less than 1 kb, at at least one given site; when the inserted fragment at the given site is a large fragment larger than 500 bp, the homologous arm length is 100-1500 bp; when base substitution, repair, or small DNA fragment insertion is performed at the given site, the homologous arm length is 15-1000 bp. Optionally, it further includes: The cells that have undergone the gene editing are subjected to at least one repeated editing, wherein the repeated editing targets the same or different given sites for the same or different gene editing. Optionally, the gene editing includes simultaneously introducing point mutations, gene insertions, deletions, or substitutions at multiple given sites without causing adverse events due to DNA double-strand cutting, such as additional chromosomal translocations or inversions. Optionally, the plurality of given sites are located on at least two different chromosomes.

30. A method for long-fragment gene editing in cells, characterized in that, include: Contact the composition according to any one of claims 1 to 24 with cells to introduce nick enzyme complex and target nucleic acid molecules into the cells. The target nucleic acid molecule includes an insert fragment and homologous arms located on both sides of the insert fragment. The length of the insert fragment is not less than 100 bp, for example, not less than 200 bp, not less than 300 bp, not less than 500 bp, not less than 1 kb, and can even be as high as at least 10 kb. Optionally, the length of the homologous arm is determined based on the length of the inserted segment.

31. The method according to claim 30, characterized in that, When the inserted fragment at the given site is a large fragment larger than 500 bp in gene size, the length of the homologous arm is 100-1500 bp. When base substitution, repair, or small fragment insertion is performed at the given site, the length of the homologous arm is 15-1000 bp.

32. A method for gene therapy on a patient, characterized in that, include: Based on the patient's target genes, the sequences of the target nucleic acid molecule and the guide nucleic acid molecule were determined; The composition of any one of claims 1 to 24 is brought into contact with cells from a patient, or the method of any one of claims 25 to 31 is used to introduce into the cells encoding a nick enzyme complex and a target nucleic acid molecule to perform gene editing at at least one given site in the cells.

33. The method according to claim 32, characterized in that, Further includes: The cells that have undergone the gene editing are subjected to at least one repeated editing, wherein the repeated editing is performed on the same or different given sites, and the same or different gene editing is performed.

34. The method according to claim 32, characterized in that, The contact is performed in vivo or in vitro; optionally, the cells are derived from a patient or allogeneic organism, and are introduced into the patient after contact with the composition according to any one of claims 1 to 24.

35. The method according to claim 32, characterized in that, The patient suffers from a gene mutation-related disease.

36. The method according to claim 32, characterized in that, The patient's cells were non-germ cells; Optionally, the given site is a disease-related gene, a cell-engineered editing site, or a safe harbor site; Optionally, the cells include patient-derived or allogeneic stem cells, somatic cells, cancer cells, and immune cells; Optionally, the given site includes at least one of the following: safe harbor site, TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, CIITA, BATF, CII-TA, HALE, HPK1, dck, RFX interleukin-13 gene, TET2, PCD1, AAVS1, ROSA26, Cytokine-inducible SH2-containing protein, DNA methyltransferase 3alpha, Cbl-b, nuclear receptor transcription factors NR4A, Diacylglycerol kinases, adenosine A2A receptor ID3, SOX4, TGF-β receptor II, protein tyrosine phosphatase 1B, CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein, CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors, TIGIT, VISTA, CD3E, PVR / CD155, MHC, CTLA4, LAG3, TIM3, HAVCR2, CD244, CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1, REGNSE1, MED12, CCNC, PPDM1, RODH2, CD5, CD7, TGF-βreceptor, TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OTUD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, APP, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4Afamily TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1 and TIGER.

37. Use of the composition according to any one of claims 1 to 24 or the gene-edited cells by the method according to any one of claims 25 to 36 in the preparation of a medicament for the treatment and / or prevention of disease; Optionally, the genetic abnormality includes at least one of gene mutation-related diseases and autoimmune diseases.

38. The method according to any one of claims 32 to 36 or the use described in claim 37, characterized in that, The disease is either a single-gene disease or a polygenic disease.

39. The method according to any one of claims 32 to 36 or the use described in claim 37, characterized in that, The diseases mentioned include cancer, hematologic disorders, autoimmune diseases, deafness, eye diseases, metabolic diseases, AIDS, and musculoskeletal disorders. Optionally, they include sickle cell anemia and thalassemia, hereditary retinal diseases, Duchenne muscular dystrophy, ALS, myotonic dystrophy type I / II, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type I, lymphoma, autoimmune diseases, B-cell leukemia, multiple myeloma, bladder cancer, liver cancer, lung cancer, epithelial cancer, colon cancer, cervical cancer, severe combined immunodeficiency, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, systemic vasculitis, and type 1 diabetes.

40. A method for constructing gene-edited organisms, characterized in that, include: By contacting the composition of any one of claims 1 to 24 with cells, or by introducing into the cells an enzyme encoding a nickase complex and a target nucleic acid molecule via the method of any one of claims 25 to 36, gene editing is performed at at least one given site in the cells. Based on the edited cells, the gene-edited organism is obtained.

41. The method according to claim 40, characterized in that, The gene-edited organisms include genetically modified or gene-edited plants and animals and their derivatives; Optionally, the genetically modified organism includes genetically modified animals or genetically modified plants.

42. A gene-edited cell, characterized in that, The cells are obtained by gene editing of cells using the method described in any one of claims 25 to 36 or the composition described in any one of claims 1 to 24.

43. A gene-edited cell population, characterized in that, The gene-edited cell population is obtained by gene editing cells using the method described in any one of claims 25 to 36 or by using the composition described in any one of claims 1 to 24.

44. A genetically modified organism, characterized in that, It is obtained by the method of claim 40 or 41.

45. A method for constructing recombinant immune cells in vivo or in vitro, characterized in that, include: Cells are treated with a gene-editing composition to perform targeted gene editing at at least one given site on the cells in order to obtain edited recombinant immune cells, the gene-editing composition comprising: A nicking enzyme complex or its encoded nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; and a target nucleic acid molecule, the target nucleic acid molecule being connectable to or bound to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding. The nicking enzyme complex is adapted to generate single-stranded DNA nicks in the genome, without simultaneously cleaving double-stranded DNA, and is configured to be linked to a first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a repair template, the target nucleic acid molecule being configured to be linked to or bind to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

46. ​​The method according to claim 45, characterized in that, The cells include at least one of the following: T cells, NK cells, DNT cells, γδT cells, iPSC cells, stem cells, iPSC-derived immune cells, umbilical cord blood-derived T cells, NK T cells, virus-specific memory T cells, CD8-positive and CD4-positive naive T cells, central memory cells, Stem-like memory T cells, DC cells, microphage, TIL cells, CIK cells, autologous immune cells, allogeneic immune cells, universal immune cells, CAR-T cells, STAR-T cells, TCR-T cells, universal CAR-T cells, CAR-M cells, and CAR-NK cells.

47. The method according to claim 45, characterized in that, The recombinant immune cells are CAR-T, universal CAR-T, CAR-NK, STAR-T, CAR-M, or TCR-T; In the case of TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, and HLA-2. B、HLA-C、HLA-E、HLA-G、CIITA、BATF、CII-TA、HALE、HPK1、dck、RFX interleukin-13gene、TET2、PCD1、AAVS1、ROSA26、Cytokine-inducible SH2-containing protein、DNA methyltransferase 3alpha、Cbl-b、Nuclear receptor transcription factors NR4A、Diacylglycerol kinases、Adenosine A2A receptor ID3, SOX4, TGF-b receptor II, protein tyrosine phosphatase 1B, CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein, CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors、TIGIT、VISTA、CD3E、PVR / CD155、MHC、CTLA4、LAG3、TIM3、HAVCR2、CD 244 CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1 、REGNSE1、MED12、CCNC、PPDM1、RODH2、CD5、CD7、TGF-βreceptor、TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OT UD7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, AP At least one of the following gene editing methods is performed: P, PSEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4A family TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1, and TIGER. This involves single-site or multi-site gene editing.

48. The method according to claim 45, characterized in that, The recombinant immune cells are suitable for overexpressing or knocking down immunomodulatory factors or genes that regulate the functional activity of immune cells. Optionally, the active regulatory factors and / or immunomodulatory factors are selected from CD47, ADR, NR4A family, HLA-E, B2M-HLA-E, NK inhibitory ligand, IL-2, IL-12, IL-15, IL-18, IL21, type 1 cytokines, type 2 cytokines, engineered IL2 superkine Super2, IL33, IL25, Flt3L, IFNγ, IL-7 / CCL19, IFNα, IFNβ, TNF, NKG2A, CD16, mbIL-15, iCasp9, RQR8, HSV-TK, TGFβ-Trap, IL-23, IL-9, GM-CSF, CCL17, CCL22, XCL1, PD-1 dominant negative receptor, CTLA-4 dominant negative receptor. receptor, TIM-3DNR, LAG-3DNR, anti-PD-1 antibody fragment, anti-CTLA-4 antibody fragment, OX40L, 4-1BBL, CD80, CD86, ICOS, ICOSL, CD40L, EGFRt, CD20t, SynNotch, BiKE, Flagellin, suicide gene, tetracycline-induced expression system, miR-155sponge, c-JUN Overexpression, PD-L1, CXCL9, CXCL10, CXCL11, CD137 antigen receptor, HVEM, BTLA, VISTA, CD73, CD39, IDO1, Arginase-1, FASL, Galectin-9, TIM-1, TIM-4, SHP-1, SHP-2, SOCS1, SOCS3, T-bet, Eomes, STAT3, HIF-1α, FOXP3, Nef, BNLF2a, US2, US11, K5, NKG2D-DAP10 signaling complex, CD94 / NKG2C and regulatory miRNA, and at least one of viral-derived signaling or regulatory proteins; In the case of TRAC, B2M, CD52, HLA-I, HLA-II, HLA-A, and HLA-2. B、HLA-C、HLA-E、HLA-G、CIITA、BATF、CII-TA、HALE、HPK1、dck、RFX interleukin-13gene、TET2、PCD1、AAVS1、ROSA26、Cytokine-inducible SH2-containing protein、DNA methyltransferase 3alpha、Cbl-b、Nuclear receptor transcription factors NR4A、Diacylglycerol kinases、Adenosine A2A receptor ID3, SOX4, TGF-b receptor II, protein tyrosine phosphatase 1B, CD2, TCRab / CD3, CD5, CS1, IL-6, PR domain finger protein, CTLA-4, TRBC1 / 2, GAPDH, RASA2, ACTB, KIF11, TBP, CD70, PDCD1, Siglec receptors、TIGIT、VISTA、CD3E、PVR / CD155、MHC、CTLA4、LAG3、TIM3、HAVCR2、CD 244 CD160, IL2RA, IL7R, IL15, IL12A, CXCR4, CCR5, CXCR2, CCR7, TRBC, BCL2, FA S, BIM, GM-CSF, DGK, A2AR, PD1, CD38, CLU, NR4A2, RGS1, RGS16, RGS2, Regnase-1 、REGNSE1、MED12、CCNC、PPDM1、RODH2、CD5、CD7、TGF-βreceptor、TGF-βreceptorII, CD19, CD20, CD22, CD33, CD123, F9, MUT, SMN1, HTT, COL7A1, G6PC, CEP290, FMR1, GBA, TTR, ABCA4, OTUD 7A, CFTR, VEGFA, MYC, TP53, EGFR, ALK, JAK2, PDGFRA, KIT, KRAS, NRAS, PIK3CA, BRCA1, BRCA2, APOE, APP, P At least one of SEN1, PSEN2, SCN1A, SCN2A, CACNA1A, PCSK1, G6PD, SERPINA1, FMR2, SMAD4, NF1, NF2, VHL, TSC1, TSC2, WRN, LMNA, CDKN2A, MAPT, HTRA1, NOTCH3, PRNP, SOD2, MT-TL1, MT-ND1, TGFBR2, Rosa26, TGFBRII, NR4Afamily TCR, CD3, β2-microglobulin, FOXP3, IL2RA, HBB, LIPA, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, PCSK9, Hipp11, Col1a1, and TIGER is selected for single-site or multi-site gene editing.

49. The method according to claim 45, characterized in that, The gene editing includes: Insertion of targeted recognition CAR / TCR / STAR genes into edited cells and / or insertion of genes that regulate the function of immune cells in killing target cells and / or genes that regulate the functional activity of immune cells.

50. The method according to any one of claims 45-49, characterized in that, The genes mentioned include BCMA-CAR, CD20-CAR, CD22-CAR, CD30-CAR, CD123-CAR, CD38-CAR, CD7-CAR, ROR1-CAR, CD70-CAR, CD19-CAR, and ERBB2-CAR. CD3-CAR, mesothelin-CAR, CD7-CAR, CD138-CAR, MICA-CAR, ULBP1-CAR, ULBP6-CAR, CD33-CAR, CD123-CAR, FLT3-CAR, BCMA-CAR, CD5-CAR, NKG2D CAR, CS1-CAR, 1XX-CAR, BiTE, TCR, STAR, TCR mimic antibody gene, optimized TCRα / β chain gene, HaloTag-CAR, SNAP-TAG-CAR, synotch, CLEC12A-CAR, HER2-CAR, MSLN-CAR, PSMA-CAR, CEA-CAR, MUC1-CAR, IL13Ra2-CAR, CD171-CAR, NKG2D-CAR, GD2-CAR, EGFR / EGFRvIII-CAR, CD33-CAR, CD44v6-CAR, FLT3-CAR, CD117-CAR, CD200-CAR, CD1a-CAR, Siglec-6-CAR, B7-H3-CAR, CLDN18.At least one of 2-CAR, FOLR1-CAR, MAGE-A4-CAR, EPCAM-CAR, PD-L1-CAR, B7-H4-CAR, CAIX-CAR, CD133-CAR, uPAR-CAR, Tandem-CAR, Dual-CAR, Split-CAR, SUPRA-CAR, UniCAR, SpyTag-CAR, FITC-CAR, ON-switch CAR, OFF-switch CAR, iCasp9-CAR, TRUCK-CAR, Armored-CAR, γδT-CAR, MAIT-CAR, iNKT-CAR, IL1RAP-CAR, CSF1R-CAR, CD276-CAR, PTK7-CAR, STEAP1-CAR, ROR2-CAR, TEM8-CAR, AXL-CAR, ANXA2-CAR, PRAME-CAR, TAG72-CAR, TSPAN8-CAR, TM4SF1-CAR, BSP-CAR, NTRK1-CAR, GD3-CAR, LMP1-CAR, NY-ESO-1-CAR, c-Met-CAR, VEGFR2-CAR, CDH17-CAR, ICAM-1-CAR, switchable-CAR, separable-CAR, inhibitory-CAR, CAR-anti-PD1, masked-CAR, light-activated CAR, hypoxia-responsive CAR, FAP-CAR, Nef-CAR, and GPC3-CAR.

51. The method according to any one of claims 45-49, characterized in that, The genes that can regulate the function of immune cells in killing target cells and / or regulate the functional activity of immune cells include at least one of the following: co-stimulatory molecules, transcription factors, active regulatory factors, immunomodulatory factors, and virus-derived signaling or regulatory proteins. Optionally, the co-stimulatory molecule is selected from at least one of OX40, ICOS, and CD27; Optionally, the transcription factor is selected from at least one of BATF, TFAP4, and FOXO1; Optionally, the active regulatory factors and / or immunomodulatory factors are selected from CD47, ADR, NR4A family, HLA-E, B2M-HLA-E, NK inhibitory ligand, IL-2, IL-12, IL-15, IL-18, IL21, type 1 cytokines, type 2 cytokines, engineered IL2 superkine Super2, IL33, IL25, Flt3L, IFNγ, IL-7 / CCL19, IFNα, IFNβ, TNF, NKG2A, CD16, mbIL-15, iCasp9, RQR8, HSV-TK, TGFβ-Trap, IL-23, IL-9, GM-CSF, CCL17, CCL22, XCL1, PD-1 dominant negative receptor, CTLA-4 dominant negative receptor. receptor, TIM-3DNR, LAG-3DNR, anti-PD-1 antibody fragment, anti-CTLA-4 antibody fragment, OX40L, 4-1BBL, CD80, CD86, ICOS, ICOSL, CD40L, EGFRt, CD20t, SynNotch, BiKE, Flagellin, suicide gene, tetracycline-induced expression system, miR-155sponge, c-JUN Overexpression, PD-L1, CXCL9, CXCL10, CXCL11, CD137 antigen receptor, HVEM, BTLA, VISTA, CD73, CD39, IDO1, Arginase-1, FASL, Galectin-9, TIM-1, TIM-4, SHP-1, SHP-2, SOCS1, SOCS3, T-bet, Eomes, STAT3, HIF-1α, FOXP3, Nef, BNLF2a, US2, US11, K5, NKG2D-DAP10 signaling complex, CD94 / NKG2C, and at least one of the following: Optionally, the viral signaling or regulatory protein is selected from the Nef protein derived from HIV-1 Clade B, the BNLF2a protein derived from EBV, the US2 and US11 proteins derived from hCMV, and the K5 protein derived from KSV.

52. The method according to any one of claims 45-51, characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: Nucleic acid fragments and proteins that specifically recognize said nucleic acid fragments; Protein tag - the ligand, nucleic acid aptamer, or binding protein of the protein tag; Antigen-antibody; Optional modified non-natural amino acids-corresponding binding ligands; Optional modified non-natural bases-corresponding binding ligands; Sequence-specific DNA / RNA binding proteins and their specific binding sequences; Structure-specific DNA / RNA binding proteins and their specific binding sequences; Specific nucleic acid modifications and corresponding binding proteins, antibodies, or ligands of these modifications; Viral proteins or their functional fragments interact with corresponding cellular or soluble viral receptors; Viral proteins and their specific antibodies; Viral envelope proteins and target cell membrane lipid / protein components; Viral proteins - DNA / RNA sequences recognized by viral proteins; Small molecules are associated with protein or nucleic acid sequences that specifically recognize them.

53. The method according to any one of claims 45-52, characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: The Rep protein-PCV recognition sequence from porcine circovirus 2PCV; TALE and its specific binding sequence; ZF and its specific binding sequence; CRISPR proteins and variants and their specific recognition sequences; ParB-parS, LacI-lacO, TetR-tetO, CymR-cuO; Non-natural amino acids containing azido groups and ligands containing alkyne groups; non-natural amino acids containing azido groups and non-natural bases containing alkyne groups; non-natural amino acids containing cyclopropene groups and tetrazine compounds; non-natural amino acids containing trans-cyclooctene and S-tetrazine; non-natural amino acids containing azido groups and cyclopropane-cyclooctene; non-natural amino acids containing tetrazine groups and cyclopropene or trans-cyclooctene. AeF-DBCO; AzF-DBCO; NAEK-DBCO; HaloTag - Chlorinated alkane ligand; SnapTag - Corresponding ligand; TMP-Tag-corresponding ligand; Avidin-Biotin; Monomeric or non-monomeric or tandem streptavidin-biotin; Viral DNA / RNA binding proteins - their binding sequences or DNA / RNA components; Viral or cellular-derived DBP, SV40, NS1, ICP8, ORF, E1, E2, integrate, UL9, HMG, Rep, NS1, IE175, NP, N, NS, GAG, RRM, HLH, AT-hook motif, EBNA1 protein - its protein recognition sequence or component. SunTag-anti-GCN4 single-chain variable sequence antibody; MoonTag-gp41 nanobody.

54. The method according to any one of claims 45 to 53, characterized in that, The first recruitment component is covalently linked to the nick enzyme.

55. The method according to claim 54, characterized in that, The first recruitment component and the nick enzyme constitute a fusion protein; Optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; Optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; Optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; Optionally, the first recruitment component includes at least one Rep protein, and the second recruitment component is a Rep protein recognition sequence; Optionally, the first recruitment component includes at least one monomeric or non-monomeric streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; Optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; Optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; Optionally, the first recruitment component may include a combination of multiple individual units connected in series.

56. The method according to claim 54 or 55, characterized in that, The first recruitment component and the nick enzyme are connected by a linker.

57. The method according to claim 56, characterized in that, The linker has 15-150 amino acids.

58. The method according to claim 56, characterized in that, The linker has the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence that is at least 50% homologous to SEQ ID NO: 1, for example, at least 80% homologous, for example, at least 90% homologous, or at least 95% homologous.

59. The method according to any one of claims 45 to 53, characterized in that, The first recruitment component is non-covalently connected to the nick enzyme; Optionally, the first recruitment component includes at least one Rep protein of porcine circovirus 2 (PCV), and the second recruitment component is a PCV recognition sequence; Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component includes at least one ParB protein, and the second recruitment component is a parS sequence; Optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; Optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; Optionally, the first recruitment component includes at least one CymR protein, and the second recruitment component is a cuO sequence; Optionally, the first recruitment component includes at least one viral protein, and the second recruitment component is a viral protein recognition sequence; Optionally, the first recruitment component is a monomeric or non-monomer streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least one non-natural base, and the second recruitment component is a specific binding ligand for the non-natural base; Optionally, the first recruitment component includes at least one non-natural amino acid, and the second recruitment component is a specific binding ligand for the non-natural amino acid; Optionally, the first recruitment component includes at least one Gag protein, and the second recruitment component is an Ψ(Psi) sequence; Optionally, the first recruitment component may include a combination of multiple individual units connected in series.

60. The method according to any one of claims 45 to 59, characterized in that, The directed nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases; Optionally, the first recruitment component is directly or indirectly linked to the binding proteins of the plurality of said nucleic acid aptamers or to modifications or ligands of non-natural bases; Optionally, the directed nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases, such as at least one, at least two, or 3 to 100 of the nucleic acid aptamers or non-natural bases.

61. The method according to claim 60, characterized in that, The combination of nucleic acid aptamers and binding proteins includes at least one of the following: MS2-MCP, PP7-PCP, BoxB-lambdaN, Com-com, Streptavidin aptamer-streptavidin.

62. The method according to claim 61, characterized in that, The binding protein of the nucleic acid aptamer binds to the protein tag, the first recruitment component is linked to at least one protein tag binding fragment, and the protein tag binding protein is adapted to specifically bind to the protein tag.

63. The method according to any one of claims 45-62, characterized in that, The nicking enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment; Optionally, the first recruitment component may be directly or indirectly linked to a plurality of proteins that specifically recognize the protein tag.

64. [Detailed Rules 91, 02.12.2025] The method according to claim 63, characterized in that, The protein tag binding fragment includes at least one of the following types that specifically recognize the protein tag: scFv, Fab, Fab', F(ab')2, Fv, Nanobody, bispecific antibody, Minibodies, single-domain antibody, the protein tag binding protein; Optionally, the combination of proteins that specifically recognize the protein tag includes at least one of the following: GCN4 - recognizes scFVs that recognize GCN4, nanobodies or their respective variants; gp41 - recognizes scFVs that recognize gp41.

65. The method according to claim 63 or 64, characterized in that, The protein tag is at least one of the GCN4 sequence and the gp41 sequence.

66. The method according to claim 65, characterized in that, The nick enzyme is fused with at least one GCN4 sequence and / or gp41 sequence, and the first recruitment component is fused with an scFv or a variant thereof that specifically recognizes the GCN4 sequence and / or gp41 sequence.

67. The method according to claim 63, characterized in that, The nicking enzyme is fused with a plurality of the protein tags, for example, at least one, at least two, or 2 to 30 of the protein tags.

68. The method according to any one of claims 45 to 63, characterized in that, The first recruitment component and the second recruitment component constitute a fusion protein.

69. The method according to any one of claims 45 to 68, characterized in that, The nicking enzyme includes at least one of the following: a CRISPR-based nicking enzyme variant, a TALE or ZF nicking enzyme that cleaves single-stranded DNA, a homing endonuclease variant that cleaves only one strand of DNA, MagaTAL, ARCUS, or a complex containing the above nicking enzyme variants, and a combination of the above nicking enzyme variants and complexes with a homologous recombination repair factor. in, Optionally, the CRISPR-based nicking enzymes include SpCas9(D10A), SpCas9(H840A), and nicking enzyme variants of SaCas9, SpG Cas9, SpRY Cas9, ScCas9, Cas-CLOVER, Cas12, Cas13, CasX, CasMINI, IscB, and TnpB. Optionally, the CRISPR protein, TALE, or ZF cleavage enzyme that cuts single-stranded DNA includes CRISPR, TALE, and ZF variants based on MutH, FokI, I-AniI, I-OnuI, and I-TevI. Optionally, the nick enzyme has the protein / amino acid sequence shown in SEQ ID NO: 2; Optionally, the system has multi-core positioning signals; Optionally, the nicking enzyme comprises a combination of the above-mentioned nicking enzyme variants and homologous recombination repair factors. The protein factors that promote homologous repair include Rad51, Rad51(A190L A192L), MRN Complex(MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51(S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 proteins or variants thereof. Optionally, the target nucleic acid molecule is plasmid DNA, viral DNA / RNA, double-stranded DNA, or single-stranded DNA / RNA; Optionally, the target nucleic acid molecule contains a homologous sequence of the target site, which can be used as a template for homologous recombination / homologous repair / gene editing, and provides a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a specified site through homologous repair or for performing base substitution, repair or small fragment insertion, replacement or repair on a specific site through homologous repair. Optionally, the target nucleic acid molecule contains a homologous sequence of the target site, which can be used as a template for homologous recombination / homologous repair / gene editing, and provides a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a specified site through homologous recombination or for inserting, replacing or repairing a large fragment at a specific site through homologous recombination. Optionally, the exogenous nucleic acid sequence has homologous arms at both ends, and the homologous arms are homologous to the nucleic acid sequence of a specific region in the target genome. When the target nucleic acid molecule is used to insert a large fragment with a gene size greater than 500 bp, the length of the homologous arm is 100-1500 bp. When the target nucleic acid molecule is used for base substitution, repair or small fragment insertion, the homologous arm can also be 15-1000 bp. Optionally, the insertion site of the exogenous nucleic acid sequence is within 100 bp of the DNA single-strand nick site; Optionally, the target nucleic acid molecule may have a specific modification or a specific protein binding sequence or specific modification; Optionally, the target nucleic acid molecule is prepared by plasmid or PCR or located in a viral vector.

70. The method according to any one of claims 45-69, characterized in that, The composition further comprises: A delivery system that loads the nick enzyme complex or its encoded nucleic acid molecule and the target nucleic acid molecule.

71. The method according to any one of claims 45-69, characterized in that, The composition is suitable for use in conjunction with base editing, lead editing, and RNA editing regulation technologies.

72. The method according to any one of claims 45-71, characterized in that, The nucleic acid molecule encoding the nick enzyme complex is mounted on the plasmid; Optionally, the nucleic acid molecule encoding the nick enzyme complex is a viral vector DNA / RNA; Optionally, the nucleic acid molecule encoding the nick enzyme complex is set on RNA; Optionally, the encoding nick enzyme complex is a protein or a protein-RNA complex.

73. [Detailed Rules 91, 02.12.2025] The method according to claim 70, characterized in that, The delivery system is suitable for liposome transfection, electrotransfection, cationic polymer transfection, microinjection, lipid nanoparticle transfection, and viral vector delivery.

74. [Detailed Rules 91, 02.12.2025] The method according to claim 72, characterized in that, The mass ratio of the plasmid to the target nucleic acid molecule is (1-10):(1-10).

75. The method according to claim 72, characterized in that, The mass ratio of the RNA to the target nucleic acid molecule is (1-10):(1-10).

76. The method according to claim 72, characterized in that, The mass ratio of the protein or protein-RNA complex to the target nucleic acid molecule is (1-10):(1-10).

77. The method according to any one of claims 45-76, characterized in that, No adverse events caused by DNA double-strand cutting, such as additional chromosomal translocations or inversions, occur in the cells.

78. The method according to any one of claims 45-76, characterized in that, A cell population is treated with a gene-editing composition to perform targeted gene editing on the cell population; Optionally, it may further include: The cells that have undergone the gene editing are subjected to at least one repeated editing, wherein the repeated editing is performed on the same or different given sites with the same or different gene editing; Optionally, the gene editing includes simultaneously introducing point mutations, gene insertions, deletions, or substitutions at multiple given sites without causing adverse events due to DNA double-strand cutting, such as additional chromosomal translocations or inversions. Optionally, the plurality of given sites are located on at least two different chromosomes.

79. A recombinant immune cell, characterized in that, The recombinant immune cells are obtained by the method described in any one of claims 45 to 78.

80. A recombinant immune cell population, characterized in that, The recombinant immune cell population is obtained by the method described in any one of claims 45 to 78.

81. Use of the recombinant immune cells obtained by the method of any one of claims 45 to 78, the recombinant immune cells of claim 79, or the recombinant immune cell population of claim 80 in the preparation of a medicament for treating a disease, the disease including genetic abnormalities such as gene mutation-related diseases, cancer, and autoimmune diseases.

82. The use according to claim 81, characterized in that, The diseases mentioned include cancer, hematologic disorders, autoimmune diseases, deafness, eye diseases, metabolic diseases, AIDS, and musculoskeletal disorders. Optionally, they include sickle cell anemia and thalassemia, hereditary retinal diseases, Duchenne muscular dystrophy, amyotrophic lateral sclerosis (ALS), myotonic dystrophy type I / II, lysosomal acid lipase deficiency, phenylketonuria, familial hypercholesterolemia, hereditary tyrosinemia type I, lymphoma, autoimmune diseases, B-cell leukemia and multiple myeloma, bladder cancer, liver cancer, lung cancer, epithelial cancer, colon cancer, cervical cancer, severe combined immunodeficiency, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, systemic vasculitis, and type 1 diabetes.

83. A gene editing system, characterized in that, include: A nicking enzyme complex comprising a nicking enzyme and an optional guide nucleic acid molecule, the nicking enzyme complex being adapted to create a single-stranded nick in the genome, without simultaneously cleaving a double-stranded DNA, the nicking enzyme complex being configured to be linked to a first recruitment component; and a target nucleic acid molecule containing homologous arms at both ends and a homologous repair template, the target nucleic acid molecule being configured to be linked to a second recruitment component, the first recruitment component and the second recruitment component being adapted to form a specific binding.

84. [Detailed Rules 91, 02.12.2025] The gene editing system according to claim 83 is characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: Nucleic acid fragments and proteins that specifically recognize said nucleic acid fragments; Protein tag - the ligand, nucleic acid aptamer, or binding protein of the protein tag; Antigen-antibody; Optional modified non-natural amino acids-corresponding binding ligands; Optional modified non-natural bases-corresponding binding ligands; Sequence-specific DNA / RNA binding proteins and their specific binding sequences; Structure-specific DNA / RNA binding proteins and their specific binding sequences; Specific nucleic acid modifications and corresponding binding proteins, antibodies, or ligands of these modifications; Avidin-Biotin; Small molecules are associated with protein or nucleic acid sequences that specifically recognize them.

85. The gene editing system according to claim 83, characterized in that, The first recruitment component and the second recruitment component are adapted to form a specific binding via at least one of the following combinations: The Rep protein-PCV recognition sequence from porcine circovirus 2PCV; TALE and its specific binding sequence; ZF and its specific binding sequence; CRISPR proteins and variants and their specific recognition sequences; ParB-parS; LacI-lacO; TetR-tetO; CymR-cuO; AeF-DBCO; HaloTag - Chlorinated alkane ligand; Avidin-Biotin; Monomeric or non-monomeric or tandem monomeric streptavidin-biotin; SunTag-anti-GCN4 single-chain variable sequence antibody; MoonTag-gp41 nanobody.

86. The gene editing system according to claim 83, characterized in that, The first recruitment component is covalently linked to the nick enzyme.

87. The gene editing system according to claim 86, characterized in that, The first recruitment component and the nick enzyme constitute a fusion protein. Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component includes at least one LacI protein, and the second recruitment component is a lacO sequence; Optionally, the first recruitment component includes at least one TetR protein, and the second recruitment component is a tetR sequence; Optionally, the first recruitment component includes at least one monomeric or non-monomeric streptavidin or biotin antibody, and the second recruitment component is biotin; Optionally, the first recruitment component includes at least two tandemly linked monomeric streptavidins.

88. The gene editing system according to claim 87, characterized in that, The first recruitment component and the nick enzyme are connected by a linker.

89. The gene editing system according to claim 88, characterized in that, The linker has 15-50 amino acids.

90. The gene editing system according to claim 89, characterized in that, The linker has the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence that is at least 50% homologous to SEQ ID NO: 1, for example, at least 80% homologous, for example, at least 90% homologous, or at least 95% homologous.

91. The gene editing system according to claim 83, characterized in that, The first recruitment component is non-covalently linked to the nick enzyme. Optionally, the first recruitment component includes at least one TALE protein, and the second recruitment component is a TALE binding sequence; Optionally, the first recruitment component includes at least one ZF protein, and the second recruitment component is a ZF binding sequence; Optionally, the first recruitment component includes at least one CRISPR protein, and the second recruitment component is a CRISPR protein binding sequence; Optionally, the first recruitment component is a monomeric streptavidin or a non-monomeric streptavidin or a biotin antibody, and the second recruitment component is biotin.

92. The gene editing system according to claim 91, characterized in that, The nick enzyme is covalently linked to at least one protein tag, and the first recruitment component is linked to at least one protein tag binding fragment.

93. The gene editing system according to claim 92, characterized in that, The protein tag binding fragment includes at least one of the following types that specifically recognize the protein tag: scFv, Fab, Fab', F(ab')2, Fv, Nanobody, Bispecific antibody, Minibodies, Single-domain antibody, The protein tag binding protein.

94. The gene editing system according to claim 91, characterized in that, The protein tag is at least one of the GCN4 sequence and the gp41 sequence.

95. The gene editing system according to claim 94, characterized in that, The nick enzyme is fused with at least one GCN4 sequence and / or gp41 sequence, and the first recruitment component is fused with an scFv or a variant thereof that specifically recognizes the GCN4 sequence and / or gp41 sequence.

96. The gene editing system according to claim 92, characterized in that, The nicking enzyme is fused with a plurality of the protein tags, for example, at least 10, at least 20, or 20 to 30 of the protein tags.

97. The gene editing system according to claim 83, characterized in that, The guided nucleic acid molecule carries multiple nucleic acid aptamers or non-natural bases. Optionally, the first recruitment component is directly or indirectly linked to the binding proteins of the plurality of said nucleic acid aptamers.

98. The gene editing system according to claim 97, characterized in that, The combination of nucleic acid aptamers and binding proteins includes at least one of the following: MS2-MCP, PP7-PCP, BoxB-lambdaN, Com-com, Streptavidin aptamer-streptavidin.

99. The gene editing system according to claim 98, characterized in that, The binding protein of the nucleic acid aptamer binds to the protein tag, the first recruitment component is linked to at least one protein tag binding fragment, and the protein tag binding protein is adapted to specifically bind to the protein tag.

100. The gene editing system according to claim 83, characterized in that, The first recruitment component and the second recruitment component constitute a fusion protein.

101. The gene editing system according to claim 83, characterized in that, The nicking enzyme includes at least one of the following: a CRISPR-based nicking enzyme variant, a TALE or ZF nicking enzyme that cleaves single-stranded DNA, a homing endonuclease variant (Meganuclease) that cleaves only one strand of DNA, or a combination of the above nicking enzyme variants with a homologous recombination repair factor. in, Optionally, the CRISPR-based nicking enzymes include SpCas9(D10A), SpCas9(H840A), and nicking enzyme variants of SaCas9, SpG Cas9, SpRY Cas9, ScCas9, Cas12, Cas13, CasX, CasMINI, IscB, and TnpB. Optionally, the TALE or ZF nicking enzymes that cleave single-stranded DNA include TALE and ZF variants based on MutH, FokI, I-AniI, I-OnuI, and I-TevI. Optionally, the nick enzyme has the protein / amino acid sequence shown in SEQ ID NO: 2; Optionally, the system has multi-core positioning signals; Optionally, the nicking enzyme comprises a combination of the above-mentioned nicking enzyme variants and homologous recombination repair factors. The protein factors that promote homologous repair include Rad51, Rad51(A190L A192L), MRN Complex(MRE11-RAD50-NBS1), RPA, Palb2, EXO1, FANCD2, FANCI, ATM, ATR, Rad51(S208E_A209D), Rad52, DN1S, CtIP, BRCA1, BRCA2 proteins or variants thereof. Optionally, the target nucleic acid molecule is plasmid DNA, viral DNA, double-stranded DNA, or single-stranded DNA; Optionally, the target nucleic acid molecule serves as a template for homologous recombination repair / gene editing, providing a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a designated site through homologous recombination or for performing base substitution, repair, or small fragment insertion, replacement, or repair at a specific site through homologous recombination. Optionally, the target nucleic acid molecule serves as a template for homologous recombination repair / gene editing, providing a foreign nucleic acid sequence that provides the required genetic information. It is suitable for inserting the foreign nucleic acid sequence into a specified site through homologous recombination or for inserting, replacing, or repairing a large fragment at a specific site through homologous recombination. Optionally, the exogenous nucleic acid sequence has homologous arms at both ends, and the homologous arms are homologous to the nucleic acid sequences of specific regions in the target genome. When the target nucleic acid molecule is used to insert a large fragment with a gene size greater than 500 bp, the length of the homologous arm is 100-1500 bp. When the target nucleic acid molecule is used for base substitution, repair or small fragment insertion, the homologous arm can be as short as 15-50 bp. Optionally, the insertion site of the exogenous nucleic acid sequence is within 20 bp of the DNA single-strand nick site; Optionally, the target nucleic acid molecule carries a identifiable modification or a specific protein-binding sequence; Optionally, the target nucleic acid molecule is prepared by PCR.

102. A composition for gene editing of cells, characterized in that, include: A nicking enzyme complex or its encoded nucleic acid molecule, the nicking enzyme complex comprising a nicking enzyme, an optional guide nucleic acid molecule, and a first recruitment component; The target nucleic acid molecule carries a second recruitment component, and the first and second recruitment components are adapted to form a specific binding. Wherein, the nick enzyme complex and the target nucleic acid molecule are as defined in any one of claims 1 to 19.

103. The composition according to claim 102, characterized in that, include: A delivery system that loads the nick enzyme complex or its encoded nucleic acid molecule and the target nucleic acid molecule.

104. The composition according to claim 102, characterized in that, The composition is suitable for use in conjunction with base editing and prime editing.

105. The composition according to claim 102, characterized in that, The nucleic acid molecule encoding the nick enzyme complex is set on the plasmid.

106. The composition according to claim 103, characterized in that, The delivery system is suitable for liposome transfection, electrotransfection, cationic polymer transfection, microinjection, lipid nanoparticle transfection, and viral vector delivery.

107. The composition according to claim 105, characterized in that, The mass ratio of the plasmid to the target nucleic acid molecule is (1-10):(1-10).

108. A method for gene editing in cells, characterized in that, include: Contact the composition of any one of claims 102 to 107 with a cell to introduce a nick enzyme complex and a target nucleic acid molecule into the cell for gene editing at at least one given site in the cell.

109. The method according to claim 108, characterized in that, The given sites include: safe harbor sites, cell engineering editing sites, and disease gene sites.

110. The method according to claim 108, characterized in that, The given site includes at least one of the following: AAVS1, CCR5, Rosa26, Hipp11, Col1a1, TIGER, TRAC, B2M, PD1, HBB, LIPA, ACTB, H2B, RAB11A, FAH, HPD, PAH, DMD, SOD1, FUS, C9orf72, DMPK, NPC, LDLR, RPE65, SOX2, LDLR, and PCSK9.

111. The method according to claim 108, characterized in that, The gene editing includes introducing point mutations, gene insertions, deletions, or substitutions at at least one of the given sites.

112. The method according to claim 108, characterized in that, The gene editing includes inserting a nucleic acid fragment of not less than 100 bp, for example, not less than 1 kb, at at least one given site; when the inserted fragment at the given site is a large fragment larger than 500 bp, the homologous arm length is 100-1500 bp; when base substitution, repair, or small fragment insertion is performed at the given site, the homologous arm length is 15-50 bp. Optionally, it further includes: The cells that have undergone the gene editing are subjected to at least one repeated editing, wherein the repeated editing targets the same or different given sites for the same or different gene editing. Optionally, the gene editing includes simultaneously introducing point mutations, gene insertions, deletions, or substitutions at multiple given sites without causing adverse events due to DNA double-strand cutting, such as additional chromosomal translocations or inversions. Optionally, the plurality of given sites are located on at least two different chromosomes.

113. A method for long-fragment gene editing in cells, characterized in that, include: Contacting the composition of any one of claims 102-107 with cells to introduce a nick enzyme complex and a target nucleic acid molecule into the cells, The target nucleic acid molecule includes an insert fragment and homologous arms located on both sides of the insert fragment. The length of the insert fragment is not less than 100 bp, for example, not less than 200 bp, not less than 300 bp, not less than 500 bp, not less than 1 kb, and can even be as high as at least 10 kb. Optionally, the length of the homologous arm is determined based on the length of the inserted segment.

114. The method according to claim 113, characterized in that, When the inserted fragment at the given site is a large fragment larger than 500 bp in gene size, the length of the homologous arm is 100-1500 bp. When base substitution, repair, or small fragment insertion is performed at the given site, the length of the homologous arm is 15-50 bp.

115. A gene-edited cell, characterized in that, The cells were obtained by gene editing using the method described in any of the preceding claims, or by using the gene editing system described in any of the preceding claims, or by using the composition described in any of the preceding claims.

116. The cell according to claim 115, characterized in that, No adverse events caused by DNA double-strand cutting, such as additional chromosomal translocations or inversions, occur in the cells.

117. The cell according to claim 115, characterized in that, The cells in question are non-reproductive cells. Optionally, the given site is a gene associated with the patient's disease, or a safe harbor site. Optionally, the cells may include patient-derived or allogeneic stem cells, cancer cells, or immune cells.

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