Genome editing method and application thereof

Abstract

The present application belongs to the field of biological medicine, and relates to a genome editing method and application thereof. The present application provides a composition or system, which comprises: a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, a single-molecule target DNA RNA or a DNA polynucleotide encoding the single-molecule target DNA RNA; and a donor template comprising a nucleic acid sequence encoding a FIX protein or a variant or a functional derivative thereof; the single-molecule target DNA RNA is an RNA targeting a Transferrin gene. The composition or system provided by the present application is used for treating hemophilia, can improve the expression of the FIX protein, makes it maintain activity in blood for a longer time, thereby improving the treatment effect, reducing the dependence of patients on treatment, and enhancing the stability and effectiveness of long-term treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a genome editing method and its application. Background Technology

[0002] Hemophilia B is a hereditary clotting disorder caused by a defect in the FIX gene. Current treatment mainly relies on regular infusions of recombinant FIX protein. However, this treatment requires lifelong dependence, is expensive, and may trigger an immune response.

[0003] Exploring the permanent repair of genetic defects in patients using gene knock-in technology has long been a goal pursued by both academia and industry. Utilizing homologous recombination as a repair mechanism is an important approach. Although existing gene editing technologies have made some progress, challenges remain in further improving editing specificity and reducing off-target effects. While gene editing tools, such as CRISPR / Cas9 and TALENs, can achieve efficient gene splicing, precisely locating and modifying only the target gene without affecting other genes remains a critical issue. Off-target effects can lead to non-specific gene mutations, resulting in unpredictable biological consequences.

[0004] The repair efficiency and gene integration stability after gene editing determine the durability and reliability of therapeutic effects. Achieving stable expression of exogenous genes and regulating them to appropriate levels is a major challenge in gene editing. Overexpression may lead to toxicity or immune responses, while underexpression may fail to achieve the desired therapeutic effect. Current research largely focuses on the albumin gene; however, the complex regulatory mechanisms of albumin may lead to inconsistent or excessive expression.

[0005] Current research mainly focuses on the albumin gene, which is an effective target, but exploring other potential targets may provide new treatment strategies. Summary of the Invention

[0006] In some embodiments, the present invention provides a composition or system comprising: a DNA endonuclease or a nucleic acid encoding the DNA endonuclease; a single-molecule RNA targeting DNA or a DNA polynucleotide encoding the single-molecule RNA targeting DNA; and a donor template comprising a nucleic acid sequence encoding a FIX protein or a variant thereof or a functional derivative thereof; wherein the single-molecule RNA targeting DNA is RNA targeting the Transferrin gene.

[0007] In some embodiments, the single-molecule targeting DNA RNA is RNA that targets an exon of the Transferrin gene. In some embodiments, the single-molecule targeting DNA RNA is RNA that targets exon 17 of the Transferrin gene.

[0008] In some embodiments, the variant is FIX-padua. In some embodiments, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs.

[0009] In some embodiments, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs.

[0010] In some implementations, the exogenous protein includes eGFP protein or FIX protein.

[0011] In some implementations, the variant is FIX-padua.

[0012] In some implementations, the sequence of SpCas9 is shown in SEQ ID NO:1.

[0013] In some implementations, the sequence of SaCas9 is shown in SEQ ID NO:2.

[0014] In some implementations, the single-molecule targeting DNA RNA is sgRNA.

[0015] In some embodiments, the composition or system is for inserting a foreign FIX gene or a variant thereof or a functional derivative thereof into exon 17 of the Transferrin gene. In some embodiments, the foreign gene includes the eGFP gene or the FIX gene.

[0016] In some implementations, the variant is the FIX-padua gene.

[0017] In some embodiments, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22.

[0018] In some embodiments, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus.

[0019] In some embodiments, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus.

[0020] In some embodiments, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10.

[0021] In some embodiments, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11.

[0022] In some embodiments, the composition or system is used to treat hemophilia.

[0023] In some implementations, the hemophilia is selected from hemophilia B.

[0024] In some embodiments, the present invention provides a kit comprising: (a) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease; (b) a single-molecule RNA targeting DNA or a DNA polynucleotide encoding the single-molecule RNA targeting DNA, wherein the single-molecule RNA targeting DNA is RNA targeting the Transferrin gene; and (c) a donor template comprising a nucleic acid sequence encoding a FIX protein or a variant thereof or a functional derivative thereof.

[0025] In some implementations, (a), (b) and (c) are in the same or separate containers.

[0026] In some implementations, the variant is FIX-padua.

[0027] In some embodiments, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs.

[0028] In some implementations, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs.

[0029] In some implementations, the DNA endonuclease is selected from SpCas9 or SaCas9.

[0030] In some implementations, the sequence of SpCas9 is shown in SEQ ID NO:1.

[0031] In some implementations, the sequence of SaCas9 is as shown in SEQ ID NO:2.

[0032] In some implementations, the single-molecule targeting DNA RNA is sgRNA.

[0033] In some embodiments, the composition or system is for inserting an exogenous FIX gene or a variant thereof or a functional derivative thereof into the position of exon 17 of the Transferrin gene.

[0034] In some embodiments, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22.

[0035] In some embodiments, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus.

[0036] In some embodiments, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus.

[0037] In some embodiments, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10.

[0038] In some embodiments, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11.

[0039] In some embodiments, the composition or system is used to treat hemophilia.

[0040] In some implementations, the hemophilia is selected from hemophilia B.

[0041] In some embodiments, the present invention provides a recombinant vector composition comprising: (1) a nuclease vector comprising a DNA endonuclease or a vector encoding the DNA endonuclease; (2) a single-molecule RNA targeting DNA vector comprising a single-molecule RNA targeting DNA or a DNA polynucleotide encoding the single-molecule RNA targeting DNA; and (3) a donor template-associated vector comprising a donor template-associated vector encoding a nucleic acid sequence encoding a FIX protein or a variant thereof or a functional derivative thereof.

[0042] In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus. In some embodiments, the virus is an adeno-associated virus. In some embodiments, the viral vector is a lentiviral expression vector, a retroviral expression vector, an adenovirus expression vector, or an adeno-associated virus expression vector.

[0043] In some embodiments, the variant is FIX-padua. In some embodiments, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs.

[0044] In some implementations, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs.

[0045] In some implementations, the DNA endonuclease is selected from SpCas9 or SaCas9.

[0046] In some implementations, the sequence of SpCas9 is shown in SEQ ID NO:1.

[0047] In some implementations, the sequence of SaCas9 is shown in SEQ ID NO:2.

[0048] In some implementations, the single-molecule targeting DNA RNA is sgRNA.

[0049] In some embodiments, the recombinant vector composition is used to insert an exogenous FIX gene or a variant thereof or a functional derivative thereof into the Transferrin gene.

[0050] In some embodiments, the recombinant vector composition is used to insert an exogenous FIX gene or a variant thereof or a functional derivative thereof into the exon of the Transferrin gene.

[0051] In some embodiments, the recombinant vector composition is used to insert an exogenous FIX gene or a variant thereof or a functional derivative thereof into the position of exon 17 of the Transferrin gene.

[0052] In some implementations, the exogenous gene includes the eGFP gene or the FIX gene.

[0053] In some implementations, the variant is the FIX-padua gene.

[0054] In some embodiments, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22.

[0055] In some embodiments, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus.

[0056] In some embodiments, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus.

[0057] In some embodiments, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10.

[0058] In some embodiments, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11.

[0059] In some embodiments, the composition or system is used to treat hemophilia.

[0060] In some implementations, the hemophilia is selected from hemophilia B.

[0061] In some embodiments, the present invention provides a recombinant cell comprising any of the compositions or systems described above or the recombinant viral vector compositions described above.

[0062] In some embodiments, the recombinant cells include cells that do not express or express low levels of the FIX gene.

[0063] In some embodiments, the present invention provides a pharmaceutical composition for treating hemophilia, comprising any of the compositions or systems described above, or the recombinant vector compositions or recombinant cells described above.

[0064] In some embodiments, the present invention provides the use of any of the described compositions or systems, any of the described kits, the described recombinant vector compositions, or the described recombinant cells or the described pharmaceutical compositions in the preparation of hemophilia drugs.

[0065] The target genes are complex, and inserting all target genes at the same site does not guarantee the desired effect. Some target genes may be expressed reasonably and function reliably at these sites, while others may fail to show good results at the same sites. The inventors of this invention have discovered that inserting a foreign gene into exon 17 of the Transferrin gene can express the foreign gene at a high level. Furthermore, compared to the albumin gene, the expression level of foreign genes bound to the albumin gene is not adjustable; therefore, foreign genes such as FIX genes are often overexpressed. In contrast, the Transferrin gene and its introduced target genes are less prone to overexpression, have higher safety, are more suitable for stabilizing variable regions, and are more likely to meet patient variability. Therefore, this invention provides a valuable technical solution, offering new ideas for the expression of some foreign genes and new approaches for the treatment of some diseases.

[0066] In some implementations, the hemophilia is hemophilia B.

[0067] In some embodiments, the present invention provides a method for modifying target DNA, comprising contacting the target DNA with a complex, the complex comprising: (a) A DNA endonuclease or a nucleic acid encoding the DNA endonuclease, (b) A single-molecule RNA targeting DNA or a DNA polynucleotide encoding the single-molecule RNA targeting DNA, and (c) A donor template comprising a nucleic acid sequence encoding a FIX protein or a variant thereof or a functional derivative thereof; the contact is in vitro or in vitro in cells; the target DNA is the Transferrin gene; the modification is to contact the complex with the Transferrin gene such that the FIX gene or a variant thereof or a functional derivative thereof is inserted into the Transferrin gene, thereby enabling the expression of Transferrin and the expression of FIX or a functional derivative thereof.

[0068] In some embodiments, the single-molecule targeting DNA RNA is an RNA that targets the exons of the Transferrin gene.

[0069] In some embodiments, the single-molecule targeting DNA RNA is an RNA that targets exon 17 of the Transferrin gene.

[0070] In some implementations, the variant is FIX-padua.

[0071] In some embodiments, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs.

[0072] In some implementations, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs.

[0073] In some implementations, the DNA endonuclease is selected from SpCas9 or SaCas9.

[0074] In some embodiments, the sequence of SpCas9 is as shown in SEQ ID NO:1; In some implementations, the sequence of SaCas9 is shown in SEQ ID NO:2.

[0075] In some implementations, the single-molecule targeting DNA RNA is sgRNA.

[0076] In some embodiments, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22.

[0077] In some embodiments, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus. In some embodiments, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus.

[0078] In some embodiments, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10. In some embodiments, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11.

[0079] In some embodiments, the present invention provides an sgRNA selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22. Attached Figure Description

[0080] Figure 1 This illustration demonstrates the expression of eGFP mRNA when eGFP is inserted into the last exon of the transferrin gene.

[0081] Figure 2 This illustration demonstrates the expression of hFIX mRNA when the human FIX gene is inserted into the last exon of the transferrin gene.

[0082] Figure 3 Immunohistochemical imaging results demonstrating the location of eGFP insertion into the last exon of the transferrin gene in wild-type mice (4 weeks after injection).

[0083] Figure 4 A diagram illustrating the quantitative results of immunohistochemical assays to verify the position of eGFP insertion into the last exon of the transferrin gene in wild-type mice (4 weeks after injection).

[0084] Figure 5The illustration shows the expression of eGFP mRNA in wild-type mice after 4 weeks of injection, verifying the insertion of eGFP into the last exon of the transferrin gene.

[0085] Figure 6 The illustration shows the results of eGFP immunohistochemical imaging in wild-type mice (4 and 8 weeks after injection) to verify the position of eGFP insertion into the last exon of the transferrin gene.

[0086] Figure 7 The illustration shows the immunohistochemical results of verifying the insertion of hFIX into the last exon of the transferrin gene in FIX knockout mice (4 weeks after injection).

[0087] Figure 8 This diagram illustrates the quantitative results of immunohistochemical assays used to verify the insertion of hFIX into the last exon of the transferrin gene in FIX knockout mice (4 weeks after injection).

[0088] Figure 9 The illustration shows the hFIX mRNA expression in FIX knockout mice after 4 weeks of injection, verifying the insertion of hFIX into the last exon of the transferrin gene.

[0089] Figure 10 A schematic diagram showing the cleavage sites corresponding to gRNA4-gRNA8.

[0090] Figure 11 This illustrates the expression of eGFP mRNA at relevant positions in the transferrin gene where eGFP is inserted.

[0091] Figure 12 The illustration shows the Western blot results of eGFP protein and corresponding actin control protein expression in liver tissue from wild-type mice, verifying the insertion of eGFP into the last exon of the transferrin gene. Detailed Implementation

[0092] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0093] Unless otherwise defined, all technical and scientific terms used herein are defined identically to those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention; preferred methods and materials are described in the detailed embodiments.

[0094] As used herein, the terms “comprising,” “having,” “containing,” and “including,” as well as other similar forms and their grammatical equivalents, are intended to be semantically equivalent and open-ended, because one or more items following any of these words do not imply an exhaustive list of those items or that they are limited to only the listed items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., containing only components A, B, and C), or may contain not only components A, B, and C, but may also include one or more other components. Therefore, it is intended and understood that “comprising” and its similar forms and their grammatical equivalents encompass disclosures of embodiments “consistently consisting of” or “comprises of.”

[0095] As used herein and in the appended claims, the singular forms “a / an,” “a / an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a method” includes multiple such methods, and reference to “the segment” includes reference to one or more segments and their equivalents known to those skilled in the art, and so on.

[0096] Furthermore, unless otherwise stated, the use of "or" means "and / or". Similarly, "contains" and "includes" are interchangeable and are not intended to be restrictive.

[0097] It should be further understood that, when the term “comprising” is used to describe various embodiments, those skilled in the art will understand that, in certain specific circumstances, the language “substantially consisting of” or “consisting of” may be used instead to describe the embodiments.

[0098] It should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, and these methods are subject to change. The terminology used herein is for the purpose of describing specific embodiments or aspects only and is not intended to limit the scope of this disclosure.

[0099] Unless otherwise specified, the experimental methods used in the following embodiments of the present invention are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0100] In this application, the term "modification" generally refers to the alteration of natural or synthetic components. Modification can include modifications to bases, nucleosides, sugars, and internucleotide bonds; it can include both chemical and non-chemical modifications.

[0101] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and a variety of other polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attachable mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the transcription or expression of coding nucleotide sequences to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0102] Some vectors, referred to herein as "recombinant expression vectors" (or simply "expression vectors"), are vectors, plasmids, or media designed to enable the expression of an inserted nucleic acid sequence after transformation into a host. Generally, expression vectors used in recombinant DNA technology are often in plasmid form. The terms "plasmid" and "vector" are used interchangeably in this specification because plasmids are the most commonly used form of vector. However, this invention includes other forms of such expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve an equivalent function.

[0103] The term "plasmid" refers to extrachromosomal elements that often carry genes that are not part of the cell's central metabolism and are often in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome-integrated sequences, bacteriophages, or nucleotide sequences from any source, linear, circular, or supercoiled, single-stranded or double-stranded DNA or RNA, many of which have been ligated or recombined into a unique structure capable of introducing promoter fragments and DNA sequences targeting selected gene products, along with suitable 3' untranslated sequences, into the cell.

[0104] The term "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by cells. When exogenous or heterologous RNA or DNA has been introduced into a cell, the cell is "transfected" by such RNA or DNA. When the transfected RNA or DNA affects phenotypic changes, the cell is "transformed" by the exogenous or heterologous RNA or DNA. Transforming RNA or DNA can be integrated (covalently linked) into the chromosomal DNA that makes up the cell's genome.

[0105] The term "homology" or "homogeneity" refers to two sequences, such as nucleotide or amino acid sequences, that, upon optimal alignment and comparison, share at least about 75% of their nucleotides or amino acids, at least about 80% of their nucleotides or amino acids, or at least about 90-95% of their nucleotides or amino acids, for example, more than 97% of their nucleotides or amino acids. As used herein, homologous arms and target sites (i.e., homologous genomic regions) are complementary to each other when two regions share a sufficient level of sequence identity, thus acting as substrates for homologous recombination reactions. "Homology" refers to the identical or shared sequence identity of a DNA sequence with a corresponding or "complementary" sequence. Homologous complementary regions between homologous arms and complementary target sites can have any length sufficient to promote homologous recombination at the cleavage recognition site. Thus, homologous arms have sufficient homology with their corresponding target sites within the cell's genome to allow for homologous recombination. For ease of reference, homologous arms are referred to herein as 5' homologous arms and 3' homologous arms. In one particular embodiment, the sum of the 5' and 3' homologous arms is at least 10 kb, or the sum of the 5' and 3' homologous arms is at least about 16 kb to about 100 kb or about 30 kb to about 100 kb. In other embodiments, the sum of the 5' and 3' homologous arms is about 10 kb to about 150 kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb, about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb to about 75 kb, or about 30 kb to about 150 kb. b. Approximately 30kb - approximately 100kb, approximately 30kb - approximately 75kb, approximately 40kb - approximately 150kb, approximately 40kb - approximately 100kb, approximately 40kb - approximately 75kb, approximately 50kb - approximately 150kb, approximately 50kb - approximately 100kb, or approximately 50kb - approximately 75kb, approximately 10kb - approximately 30kb, approximately 20kb - approximately 40kb, approximately 40kb - approximately 60kb, approximately 60kb - approximately 80kb, approximately 80kb - approximately 100kb, approximately 100kb - approximately 120kb, or approximately 120kb - approximately 150kb.

[0106] In this article, SaCas9 / gRNA3 is the same as SaCas9-gRNA3, and so on.

[0107] SpCas9, SaCas9, and TALENs DNA were synthesized by GenScript Biotech Ltd. AAV8 was sourced from Newp Biotech Ltd. The eGFP sequence (Gene. 1996;173(1 Spec No):33-8. doi: 10.1016 / 0378-1119(95)00685-0.) and hFIX sequence (N. Engl. J. Med. 2009;361:1671-1675) were derived from published articles and synthesized by GenScript Biotech Ltd. In the embodiments described herein, hFIX refers to FIX-Padua. The relevant sequences are detailed in Table 1 below.

[0108] Table 1 Sequence Information In Table 1, SEQ ID NO:5-7 and SEQ ID NO:18-22 are the RNA complementary sequences of the corresponding target DNA.

[0109] Example 1: In vitro experiment to verify the expression of eGFP after insertion into the last exon of the transferrin gene. 1. Experimental Methods 1.1 Cloning and Construction of Vectors The cloning and construction of the vector were performed using the CloneExpress cloning kit. pUC57-AmpR plasmid DNA was extracted according to the manufacturer's instructions and the concentration was adjusted to 500 ng / μL. Restriction endonucleases provided by New England BioLabs (NEB) were added, and the mixture was incubated in a 37°C water bath for 2 hours to ensure complete digestion. Next, a DNA fragment containing the synthesized eGFP sequence (sequences shown in SEQ ID NO:4, SEQ ID NO:8; SEQ ID NO:9) containing both 5' and 3' homologous sequences, or a DNA fragment containing the CMV promoter SpCas9 and U6 promoter gRNA1 sequence (sequences shown in SEQ ID NO:1 and SEQ ID NO:5), or a DNA fragment containing the CMV promoter SaCas9 and U6 promoter gRNA2 sequence (sequences shown in SEQ ID NO:2 and SEQ ID NO:6) was added. The mixture was ligated into the plasmid using T4 DNA ligase (NEB) at 16°C for 16 hours to obtain the recombinant plasmid carrying the target genes. The recombinant plasmid was transformed into competent *E. coli* DH5α using a heat shock method (42°C for 45 seconds), followed immediately by placing on ice for 2 minutes. Then, 500 μL of LB medium was added, and the culture was incubated at 37°C with shaking at 220 rpm for 1 hour. An appropriate amount of culture was plated onto LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated at 37°C for 12–16 hours. Single colonies were picked and inoculated into 5 mL of LB agar (containing 100 μg / mL Amp), and incubated at 37°C with shaking at 220 rpm for 12–16 hours. Plasmid extraction was then performed using the CloneExpress Plasmid Kit to obtain the plasmid carrying the target gene eGFP, or the SpCas9 / gRNA1 or SaCas9 / gRNA2 plasmid required for subsequent experiments.

[0110] 1.2 Production of AAV8 AAV8 was produced using HEK293T cells cultured in DMEM basal medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (PS) at a density of 1 × 10⁻⁶ cells / year in a humidified incubator at 37°C and 5% CO₂. 7 Cells / 150 mm cell culture dish.

[0111] AAV8 was prepared using a 3-plasmid transfection method: The pAAV plasmid (carrying the target eGFP gene, or SpCas9 / gRNA1, or SaCas9 / gRNA2), pHelper plasmid, and pRep-Cap plasmid (AAV8 serotype) prepared in step 1.1 were selected. Transfection was performed using PEI (25 kDa, linear, Polysciences) at a PEI:plasmid mass ratio of 3:1. For transfection, 500 μg of a 1:1:1 mass ratio of pAAV:pHelper:pRep-Cap plasmid DNA was added to 2 mL of Opti-MEM and mixed thoroughly to obtain the plasmid solution. 1.5 mg of PEI was weighed and added to 2 mL of Opti-MEM, and after complete dissolution, the PEI solution was obtained.

[0112] The plasmid solution was mixed with the PEI solution and incubated at room temperature for 10 minutes. The mixture was then evenly added to a culture dish containing HEK293T cells and cultured at 37°C and 5% CO2 for 72 hours. After culturing, 10 mL of PBS + 0.001% Pluronic F-68 polyol solution (Thermo Fisher) was added, and the mixture was subjected to three freeze-thaw cycles (5 min in liquid nitrogen – 5 min at 37°C) to ensure complete lysis. The lysate was then purified using a Backman ultracentrifuge at 50,000 × g for 24 hours using a Beckman Coulter SW41Ti rotor. After centrifugation, the fraction containing AAV8 particles was carefully removed and dialyzed against PBS using an Amicon Ultra-15 100 kDa cutoff membrane at 4,000 × g and 4°C. After complete dialysis, recombinant AAV8-eGFP, AAV8-SpCas9 / gRNA1, or AAV8-SaCas9 / gRNA2 viral particles were obtained for subsequent experiments. Quantification was performed using qPCR, ITR region-specific primers, and the standard curve method.

[0113] 1.3 In vitro hepatocyte gene editing experiment In vitro hepatocyte gene editing experiments were conducted using HepG2 (human hepatocellular carcinoma cell line) under the following conditions: DMEM + 10% FBS + 1% PS, 37°C, 5% CO2 humidified incubator. The cell seeding density was 3 × 10⁶ cells / year. 5Cells / well (12-well plate), 1 mL of culture medium per well. HepG2 cells adhered to the plate for 16 hours before transfection. Transfection reagent was Lipo3000 (ThermoFisher). 500 ng / well of plasmid SpCas9 / gRNA1, or SaCas9 / gRNA2, or TALEN plasmid was used.

[0114] For transfection, mix 2 μL of Lipo3000 with 50 μL of Opti-MEM and incubate at room temperature for 5 min. Then, add 500 ng of each of the above-mentioned SpCas9 / gRNA1, SaCas9 / gRNA2, or TALEN1 plasmid DNA to 50 μL of Opti-MEM and incubate at room temperature for 5 min. Combine the two mixtures, mix thoroughly, and incubate at room temperature for 10 min. Add the mixture evenly to HepG2 cell culture dishes (containing DMEM + 10% FBS) and incubate with HepG2 cells at 37°C for 6 hours. Then, dilute AAV8-eGFP virus in DMEM + 10% FBS and infect the HepG2 cells with the AAV8-eGFP MOI: 1 × 10⁻⁶. 5 Viral genome (vg) / cell, continue incubation at 37°C, 5% CO2 humidifier for 48 hours.

[0115] The preparation method of TALEN1 (sequences shown in SEQ ID NO:16 and SEQ ID NO:17) is the same as step 1.1 above.

[0116] 1.4 qPCR analysis to quantify eGFP mRNA expression For qPCR analysis of eGFP mRNA expression, TRIzol (Invitrogen) was used. 500 μL of TRIzol was added to lyse HepG2 cells and incubated at room temperature for 5 min. 100 μL of chloroform was added, the mixture was vigorously vortexed for 15 s, and incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 × g at 4°C for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, and the mixture was incubated at -20°C for 10 min. The mixture was then centrifuged at 12,000 × g at 4°C for 10 min, the supernatant was discarded, and the cells were washed twice with 75% ethanol. The RNA was dissolved in 20 μL of Nase-free water and incubated at 37°C for 5 min. This solution was then used for cDNA synthesis. The cDNA synthesis reaction system consisted of 1 μg RNA + 4 μL 5× RT Buffer + 1 μL Random Primer + 1 μL dNTP Mix + 0.5 μL M-MLV reverse transcriptase + DEPC water to a final volume of 20 μL. Reaction conditions: 25°C for 10 min, 42°C for 50 min, 70°C for 10 min. The synthesized cDNA was stored at -20°C. The qPCR system consisted of 10 μL SYBR Green + 2 μL cDNA + 0.5 μL of each primer (SEQ ID NO:12:5'-AGTCCGCCCTGAGCAAAGA-3'(eGFP-F) and SEQ ID NO:13:5'-TCCAGCAGGACCATGTGATC-3' (eGFP-R)) (10 μM) + ddH2O to a final volume of 20 μL. Reaction conditions: 95°C for 5 min, 95°C for 30 s, 61°C for 30 s (38 cycles), 72°C for 10 min. The internal reference gene was β-actin, and the relative gene expression level was calculated using the ΔΔCt method.

[0117] The groups include: (a) Control group: HepG2 cells were transfected using only the Lipo3000 transfection reagent.

[0118] (b) AAV-eGFP group: HepG2 cells were infected with only AAV8-eGFP.

[0119] (c) AAV-eGFP+SpCas9 / gRNA1 group: HepG2 cells were infected with AAV8-eGFP and simultaneously transfected with SpCas9 / gRNA1 plasmid.

[0120] (d) AAV-eGFP+SaCas9 / gRNA2 group: HepG2 cells were infected with AAV8-eGFP and simultaneously transfected with SaCas9 / gRNA2 plasmid.

[0121] (e) AAV-eGFP + TALEN1 group: HepG2 cells were infected with AAV8-eGFP and simultaneously transfected with TALEN1 plasmid.

[0122] 2. Experimental Results The results are shown in Table 2 and Figure 1 As shown, the experimental results demonstrate that this invention successfully verified the position of eGFP inserted into the last exon of the transferrin gene, which can effectively express the GFP gene.

[0123] Table 2 Example 2: In vitro experimental verification of hFIX gene expression after insertion into the last exon of the transferrin gene. 1. Experimental Methods 1.1 Cloning and Construction of Vectors The cloning and construction of the vector were performed using the CloneExpress cloning kit. pUC57-AmpR plasmid DNA was extracted according to the manufacturer's instructions and the concentration was adjusted to 500 ng / μL. Restriction endonucleases provided by New England BioLabs (NEB) were added, and the mixture was incubated in a 37°C water bath for 2 hours to ensure complete digestion. Next, a synthesized DNA fragment containing 5' and 3' homologous sequences (sequences shown in SEQ ID NO:3, SEQ ID NO:8; SEQ ID NO:9), or a DNA fragment containing the CMV promoter SpCas9 and U6 promoter gRNA1 (sequences shown in SEQ ID NO:1 and SEQ ID NO:5), or a DNA fragment containing the CMV promoter SaCas9 and U6 promoter gRNA2 (sequences shown in SEQ ID NO:2 and SEQ ID NO:6) was added. The mixture was ligated into the plasmid using T4 DNA ligase (NEB) at 16°C for 16 hours to obtain the recombinant plasmid carrying the target genes. The recombinant plasmid was transformed into competent *E. coli* DH5α using a heat shock method (42°C for 45 seconds), followed immediately by placing on ice for 2 minutes. Then, 500 μL of LB medium was added, and the culture was incubated at 37°C with shaking at 220 rpm for 1 hour. An appropriate amount of culture was plated onto LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated at 37°C for 12–16 hours. Single colonies were picked and inoculated into 5 mL of LB agar (containing 100 μg / mL Amp), and incubated at 37°C with shaking at 220 rpm for 12–16 hours. Plasmid extraction was then performed using the CloneExpress Plasmid Kit to obtain the plasmids carrying the target gene hFIX, SpCas9 / gRNA1, or SaCas9 / gRNA2 plasmids required for subsequent experiments.

[0124] 1.2 Production of AAV8 AAV8 was produced using HEK293T cells cultured in DMEM basal medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (PS) at a density of 1 × 10⁻⁶ cells / year in a humidified incubator at 37°C and 5% CO₂. 7 Cells / 150 mm cell culture dish.

[0125] AAV8 was prepared using a 3-plasmid transfection method: The pAAV plasmid (carrying the target hFIX gene, or SpCas9 / gRNA1, or SaCas9 / gRNA2), pHelper plasmid, and pRep-Cap plasmid (AAV8 serotype) prepared in step 1.1 of this embodiment were selected. Transfection was performed using PEI (25 kDa, linear, Polysciences) at a PEI:plasmid mass ratio of 3:1. For transfection, 500 μg of a 1:1:1 mixture of pAAV:pHelper:pRep-Cap plasmid DNA was added to 2 mL of Opti-MEM and mixed thoroughly to obtain the plasmid solution. 1.5 mg of PEI was weighed and added to 2 mL of Opti-MEM, and after complete dissolution, a PEI solution was obtained.

[0126] The plasmid solution was mixed with the PEI solution and incubated at room temperature for 10 minutes. The mixture was then evenly added to a culture dish containing HEK293T cells and cultured at 37°C and 5% CO2 for 72 hours. After culturing, 10 mL of PBS + 0.001% Pluronic F-68 polyol solution (Thermo Fisher) was added, and the mixture was subjected to three freeze-thaw cycles (5 min in liquid nitrogen – 5 min at 37°C) to ensure complete lysis. The lysate was then purified using an ultracentrifuge (Backman) with a gradient centrifugation using iodixanol on a Beckman Coulter SW41Ti rotor at 50,000 × g for 24 hours. After centrifugation, the fraction containing AAV8 particles was carefully removed and dialyzed against PBS using an Amicon Ultra-15 100 kDa cutoff membrane at 4,000 × g and 4°C. After dialysis was completed, recombinant AAV8-hFIX, AAV8-SpCas9 / gRNA1, or AAV8-SaCas9 / gRNA2 viral particles were obtained for subsequent experiments. Quantification was performed using qPCR, ITR region-specific primers, and the standard curve method.

[0127] 1.3 In vitro hepatocyte gene editing experiment In vitro hepatocyte gene editing experiments were conducted using HepG2 (human hepatocellular carcinoma cell line) under the following conditions: DMEM + 10% FBS + 1% PS, 37°C, 5% CO2 humidified incubator. The cell seeding density was 3 × 10⁶ cells / year. 5Cells / well (12-well plate), 1 mL of culture medium per well. HepG2 cells adhered to the plate for 16 hours before transfection. Transfection reagent was Lipo3000 (Thermo Fisher). 500 ng / well of plasmid SpCas9 / gRNA1, or SaCas9 / gRNA2, or TALEN plasmid was used.

[0128] For transfection, mix 2 μL of Lipo3000 with 50 μL of Opti-MEM and incubate at room temperature for 5 min. Then, add 500 ng of each of the above-mentioned SpCas9 / gRNA1, SaCas9 / gRNA2, or TALEN1 plasmid DNA to 50 μL of Opti-MEM and incubate at room temperature for 5 min. Combine the two mixtures, mix thoroughly, and incubate at room temperature for 10 min. Add the mixture evenly to HepG2 cell culture dishes (containing DMEM + 10% FBS) and incubate with HepG2 cells at 37°C for 6 hours. Then, dilute AAV8-hFIX virus in DMEM + 10% FBS and infect the HepG2 cells with the AAV8-hFIX MOI: 1 × 10⁻⁶. 5 Viral genome (vg) / cell was collected and cultured in a humidified incubator at 37°C for 48 hours. TALEN1 (sequences shown in SEQ ID NO:16 and SEQ ID NO:17) was obtained using the same method as in Example 1.

[0129] 1.4 qPCR analysis to quantify hFIX mRNA expression For qPCR analysis of hFIX mRNA expression, TRIzol (Invitrogen) was used. 500 μL of TRIzol was added to lyse HepG2 cells and incubated at room temperature for 5 min. 100 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s, then incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 × g at 4°C for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, and the mixture was incubated at -20°C for 10 min. The mixture was then centrifuged at 12,000 × g at 4°C for 10 min, the supernatant was discarded, and the cells were washed twice with 75% ethanol. The RNA was dissolved in 20 μL of Nase-free water and incubated at 37°C for 5 min. This solution was then used for cDNA synthesis. The cDNA synthesis reaction system consisted of 1 μg RNA + 4 μL 5× RT Buffer + 1 μL Random Primer + 1 μL dNTP Mix + 0.5 μL M-MLV reverse transcriptase + DEPC water to a final volume of 20 μL. Reaction conditions: 25°C for 10 min, 42°C for 50 min, 70°C for 10 min. The synthesized cDNA was stored at -20°C. The qPCR system consisted of 10 μL SYBR Green + 2 μL cDNA + 0.5 μL of each primer (SEQ ID NO:14 5'-GGTGGAGAAGATGCCAAACCAG-3' (hFIX-F) and SEQ ID NO:15 5'-CAACACAGTGGGCAGCAGTTAC-3' (hFIX-R)) (10 μM) + ddH2O to a final volume of 20 μL. Reaction conditions: 95°C for 5 min, 95°C for 30 s, 61°C for 30 s (38 cycles), 72°C for 10 min. The internal reference gene was β-actin, and the relative gene expression level was calculated using the ΔΔCt method.

[0130] The groups include: (a) Control group: HepG2 cells were transfected using only the Lipo3000 transfection reagent.

[0131] (b) AAV-hFIX group: HepG2 cells were infected with AAV8-hFIX only.

[0132] (c) AAV-hFIX + SpCas9 / gRNA1 group: HepG2 cells were infected with AAV8-hFIX and simultaneously transfected with SpCas9 / gRNA1 plasmid.

[0133] (d) AAV-hFIX + SaCas9 / gRNA2 group: HepG2 cells were infected with AAV8-hFIX and simultaneously transfected with SaCas9 / gRNA2 plasmid.

[0134] (e) AAV-hFIX + TALEN1 group: HepG2 cells were infected with AAV8-hFIX and simultaneously transfected with TALEN1 plasmid.

[0135] 2. Experimental Results The results are shown in Table 3 and Figure 2 As shown, the experimental results demonstrate that this invention successfully verified the position of hFIX inserted into the last exon of the transferrin gene, which enables the hFIX gene to be expressed well.

[0136] Table 3 Example 3: In vivo experiments to verify the expression of eGFP inserted into the last exon of the transferrin gene. 1. Experimental Materials Mice: C57BL / 6 wild-type newborn mice, day 2, sex-balanced, weighing approximately 1-2 grams.

[0137] 2. Experimental Methods 2.1 Cloning and Construction of Vectors The vector was cloned and constructed using the CloneExpress cloning kit. pUC57-AmpR plasmid DNA was extracted according to the manufacturer's instructions and the concentration was adjusted to 500 ng / μL. Restriction endonucleases provided by New England BioLabs (NEB) were added, and the mixture was incubated at 37°C for 2 hours to ensure complete digestion. Next, a synthesized DNA fragment containing both 5' and 3' homologous sequences of eGFP (sequences shown in SEQ ID NO:4, SEQ ID NO:10; SEQ ID NO:11), or a DNA fragment containing the CMV promoter SaCas9 and the U6 promoter gRNA3 sequence (sequences shown in SEQ ID NO:2 and SEQ ID NO:7) was added. The mixture was ligated into the plasmid using T4 DNA ligase (NEB) at 16°C for 16 hours to obtain recombinant plasmids carrying the target genes. The recombinant plasmid was transformed into competent *E. coli* DH5α using a heat shock method (42°C for 45 seconds), followed immediately by placing on ice for 2 minutes. Then, 500 μL of LB medium was added, and the culture was incubated at 37°C with shaking at 220 rpm for 1 hour. An appropriate amount of culture was plated onto LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated at 37°C for 12–16 hours. Single colonies were picked and inoculated into 5 mL of LB agar (containing 100 μg / mL Amp), and incubated at 37°C with shaking at 220 rpm for 12–16 hours. Plasmid extraction was then performed using the CloneExpress Plasmid Kit to obtain either the plasmid carrying the target gene eGFP or the SaCas9 / gRNA3 plasmid required for subsequent experiments.

[0138] 2.2 Production of AAV8 AAV8 was produced using HEK293T cells cultured in DMEM basal medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (PS) at a density of 1 × 10⁻⁶ cells / year in a humidified incubator at 37°C and 5% CO₂. 7 Cells / 150 mm cell culture dish.

[0139] AAV8 was prepared using a 3-plasmid transfection method: The pAAV plasmid (carrying the target eGFP gene, or SaCas9 / gRNA3), pHelper plasmid, and pRep-Cap plasmid (AAV8 serotype) prepared above were selected. Transfection was performed using PEI (25 kDa, linear, Polysciences) at a PEI:plasmid mass ratio of 3:1. For transfection, 500 μg of a 1:1:1 mixture of pAAV:pHelper:pRep-Cap plasmid DNA was added to 2 mL of Opti-MEM and mixed thoroughly to obtain the plasmid solution. 1.5 mg of PEI was weighed and added to 2 mL of Opti-MEM, and after complete dissolution, the PEI solution was obtained.

[0140] The plasmid solution was mixed with PEI solution and incubated at room temperature for 10 minutes. The mixture was then evenly added to a culture dish containing HEK293T cells and cultured at 37°C with 5% CO2 for 72 hours. After culturing, 10 mL of PBS + 0.001% Pluronic F-68 polyol solution (Thermo Fisher) was added, and the mixture was subjected to three freeze-thaw cycles (5 min in liquid nitrogen – 5 min at 37°C) to ensure complete lysis. The lysate was then purified using an ultracentrifuge (Backman) with a gradient centrifugation using iodixanol on a Beckman Coulter SW41Ti rotor at 50,000 × g for 24 hours. After centrifugation, the fraction containing AAV8 particles was carefully removed and dialyzed against PBS using an Amicon Ultra-15 100 kDa cutoff membrane at 4,000 × g and 4°C. After complete dialysis, recombinant AAV8-eGFP and AAV8-SaCas9 / gRNA3 viral particles were obtained for subsequent experiments. Quantification was performed using qPCR, ITR region-specific primers, and the standard curve method.

[0141] 2.3 Injection in mice: On day 2 after birth, newborn mice were administered the drug via intravenous injection (infraorbital vein). Each mouse was injected with the prepared AAV8-eGFP and AAV8-SaCas9 / gRNA3 viral particles (dose: 5 × 10¹³ vg / kg). The viral particles, diluted in PBS, were precisely injected into each mouse using a microsyringe (10 μL, 30G), ensuring correct injection site. After injection, the mice were observed for recovery, ensuring no abnormal reactions. Body temperature and food supply were maintained, and overstimulation was avoided. The mice were continuously observed at week 4 and week 8.

[0142] 2.4 qPCR analysis to quantify eGFP mRNA expression At the end of the experiment, animals were euthanized according to standard procedures, and liver tissue was collected. For qPCR analysis of eGFP mRNA expression, TRIzol (Invitrogen) was used. 500 μL of TRIzol was added to lyse 50 mg of liver tissue sample, and incubated at room temperature for 5 min. 100 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s, then incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 × g at 4°C for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, and the mixture was incubated at -20°C for 10 min. The mixture was then centrifuged at 12,000 × g at 4°C for 10 min, the supernatant was discarded, and the sample was washed twice with 75% ethanol. The RNA was dissolved in 20 μL of RNase-free water and incubated at 37°C for 5 min. This solution was then used for cDNA synthesis. The cDNA synthesis reaction system consisted of 1 μg RNA + 4 μL 5× RT Buffer + 1 μL Random Primer + 1 μL dNTP Mix + 0.5 μL M-MLV reverse transcriptase + DEPC water to a final volume of 20 μL. Reaction conditions: 25°C for 10 min, 42°C for 50 min, 70°C for 10 min. The synthesized cDNA was stored at -20°C. The qPCR system consisted of 10 μL SYBR Green + 2 μL cDNA + 0.5 μL each primer (5'-AGTCCGCCCTGAGCAAAGA-3'(eGFP-F) and 5'-TCCAGCAGGACCATGTGATC-3'(eGFP-R)) (10 μM) + ddH2O to a final volume of 20 μL. Reaction conditions: 95°C for 5 min, 95°C for 30 s, 61°C for 30 s (38 cycles), 72°C for 10 min. The internal reference gene was β-actin, and the relative gene expression level was calculated using the ΔΔCt method for data analysis.

[0143] The groups include: (a) Control group: injected with PBS.

[0144] (b) AAV-eGFP group: AAV8-eGFP was injected only.

[0145] (d) AAV-eGFP + SaCas9 / gRNA3 group: Simultaneous injection of AAV8-eGFP and AAV8-SaCas9 / gRNA3 virus.

[0146] 2.5 Immunohistochemical imaging: At 4 and 8 weeks post-injection, mice were sacrificed and liver tissue was collected. Liver tissue was fixed in 4% formalin for 24 hours, followed by washing with PBS buffer. The fixed liver tissue was embedded in paraffin, cut into 5 μm thick sections, placed on slides, and subjected to anti-GFP immunohistochemical staining. The staining procedure was as follows: paraffin was removed using Xylene, followed by hydration using an alcohol gradient. Antigen retrieval: antigen retrieval was performed using citrate buffer. Blocking: nonspecific binding was blocked using PBS solution containing 5% normal goat serum. Antibody incubation: incubation was performed overnight at 4°C using rabbit anti-GFP antibody (Abcam, 1:500). Chromogenic reaction: reaction was performed using DAB chromogenic solution, followed by counterstaining with Hematoxylin. The immunohistochemical staining results were observed under an optical microscope, and GFP-positive cells were recorded.

[0147] 2. Experimental Results The results are as follows Figures 3-6 As shown in the figure, stable expression of eGFP was observed at both 4 and 8 weeks post-injection.

[0148] Example 4: In vivo experiments to verify the expression of the hFIX gene inserted into the last exon of the transferrin gene. 1. Experimental Materials Mice: Newborn mice on the second day of life, sex-balanced, weighing approximately 1-2 grams, were used in the experiment to knock out the hemophilia disease model mice using FIX knockout mice.

[0149] 2. Experimental Methods 2.1 Cloning and Construction of Vectors The cloning and construction of the vector were performed using the CloneExpress cloning kit. pUC57-AmpR plasmid DNA was extracted according to the manufacturer's instructions and the concentration was adjusted to 500 ng / μL. Restriction endonucleases provided by New England BioLabs (NEB) were added, and the mixture was incubated in a 37°C water bath for 2 hours to ensure complete digestion. Next, a synthesized hFIX DNA fragment containing 5' and 3' homologous sequences (sequences shown in SEQ ID NO:3, SEQ ID NO:10; SEQ ID NO:11), or a DNA fragment containing the CMV promoter SaCas9 and U6 promoter gRNA3 sequences (sequence information tables SEQ ID NO:2 and SEQ ID NO:7) was added. The mixture was ligated into the plasmid using T4 DNA ligase (NEB) at 16°C for 16 hours to obtain recombinant plasmids carrying the target genes. The recombinant plasmid was transformed into competent *E. coli* DH5α using a heat shock method (42°C for 45 seconds), followed immediately by placing on ice for 2 minutes. Then, 500 μL of LB medium was added, and the culture was incubated at 37°C with shaking at 220 rpm for 1 hour. An appropriate amount of culture was plated onto LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated at 37°C for 12–16 hours. Single colonies were picked and inoculated into 5 mL of LB agar (containing 100 μg / mL Amp), and incubated at 37°C with shaking at 220 rpm for 12–16 hours. Plasmid extraction was then performed using the CloneExpress Plasmid Kit to obtain the plasmid carrying the target gene hFIX or the SaCas9 / gRNA3 plasmid required for subsequent experiments.

[0150] 2.2 Production of AAV8 AAV8 was produced using HEK293T cells cultured in DMEM basal medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (PS) at a density of 1 × 10⁻⁶ cells / year in a humidified incubator at 37°C and 5% CO₂. 7 Cells / 150 mm cell culture dish.

[0151] AAV8 was prepared using a 3-plasmid transfection method: The pAAV plasmid (carrying the target hFIX gene, or SaCas9 / gRNA3), pHelper plasmid, and pRep-Cap plasmid (AAV8 serotype) prepared above were selected. Transfection was performed using PEI (25 kDa, linear, Polysciences) at a PEI:plasmid mass ratio of 3:1. For transfection, 500 μg of a 1:1:1 mixture of pAAV:pHelper:pRep-Cap plasmid DNA was added to 2 mL of Opti-MEM and mixed thoroughly to obtain the plasmid solution. 1.5 mg of PEI was weighed and added to 2 mL of Opti-MEM, and after complete dissolution, the PEI solution was obtained.

[0152] The plasmid solution was mixed with PEI solution and incubated at room temperature for 10 minutes. The mixture was then evenly added to a culture dish containing HEK293T cells and cultured at 37°C and 5% CO2 for 72 hours. After culturing, 10 mL of PBS + 0.001% Pluronic F-68 polyol solution (Thermo Fisher) was added, and the mixture was subjected to three freeze-thaw cycles (5 min in liquid nitrogen – 5 min at 37°C) to ensure complete lysis. The lysate was then purified using an ultracentrifuge (Backman) with a gradient centrifugation using iodixanol on a Beckman Coulter SW41Ti rotor at 50,000 × g for 24 hours. After centrifugation, the fraction containing AAV8 particles was carefully removed and dialyzed against PBS using an Amicon Ultra-15 100 kDa cutoff membrane at 4,000 × g and 4°C. After dialysis was completed, recombinant AAV8-hFIX and AAV8-SaCas9 / gRNA3 viral particles were obtained for subsequent experiments. Quantification was performed using qPCR, ITR region-specific primers, and the standard curve method.

[0153] 2.3 Injection in mice: On day 2 after birth, newborn mice were administered the drug via intravenous injection (infraorbital vein). Each mouse was injected with the prepared AAV8-hFIX and AAV8-SaCas9 / gRNA3 viral particles (dose: 5 × 10¹³ vg / kg). The viral particles, diluted in PBS, were precisely injected into each mouse using a microsyringe (10 μL, 30G), ensuring correct injection site. After injection, the mice were observed for recovery, ensuring no abnormal reactions. Body temperature and food supply were maintained, and overstimulation was avoided. Mice were observed for 4 weeks.

[0154] 2.4 qPCR analysis to quantify eGFP mRNA expression At the end of the experiment, animals were euthanized according to standard procedures, and liver tissue was collected. For qPCR analysis of eGFP mRNA expression, TRIzol (Invitrogen) was used. 500 μL of TRIzol was added to lyse 50 mg of liver tissue sample, and incubated at room temperature for 5 min. 100 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s, then incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 × g at 4°C for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, and the mixture was incubated at -20°C for 10 min. The mixture was then centrifuged at 12,000 × g at 4°C for 10 min, the supernatant was discarded, and the sample was washed twice with 75% ethanol. The RNA was dissolved in 20 μL of RNase-free water and incubated at 37°C for 5 min. This solution was then used for cDNA synthesis. The cDNA synthesis reaction system consisted of 1 μg RNA + 4 μL 5× RT Buffer + 1 μL Random Primer + 1 μL dNTP Mix + 0.5 μL M-MLV reverse transcriptase + DEPC, with water added to a final volume of 20 μL. Reaction conditions: 25°C for 10 min, 42°C for 50 min, and 70°C for 10 min. The synthesized cDNA was stored at -20°C. qPCR system: 10 μL SYBR Green + 2 μL cDNA + 0.5 μL each primer (5'-GGTGGAGAAGATGCCAAACCAG-3' (hFIX-F) and 5'-CAACACAGTGGGCAGCAGTTAC-3' (hFIX-R) (10 μM)) + ddH2O to a final volume of 20 μL. Reaction conditions: 95°C for 5 min, 95°C for 30 s, 61°C for 30 s (38 cycles), 72°C for 10 min. The internal control gene was β-actin, and the relative gene expression level was calculated using the ΔΔCt method.

[0155] The groups include: (a) Control group: injected with PBS.

[0156] (b) AAV-hFIX group: AAV8-hFIX was injected only.

[0157] (d) AAV-hFIX + SaCas9 / gRNA3 group: Simultaneous injection of AAV8-hFIX and AAV8-SaCas9 / gRNA3 virus.

[0158] 2.5 Immunohistochemical imaging: Four weeks after injection, mice were sacrificed and liver tissue was collected. The liver tissue was fixed in 4% formalin solution for 24 hours, followed by washing with PBS buffer. The fixed liver tissue was embedded in paraffin, cut into 5 μm thick sections, placed on glass slides, and subjected to anti-hFIX immunohistochemical staining. The staining procedure was as follows: paraffin was removed using Xylene, and hydration was performed using an alcohol gradient. Antigen retrieval: antigen retrieval was performed using citrate buffer. Blocking: nonspecific binding was blocked using PBS solution containing 5% normal goat serum. Antibody incubation: incubation was performed overnight at 4°C using rabbit anti-hFIX antibody (Abcam, 1:500). Colorimetric reaction: reaction was performed using DAB chromogenic solution, and counterstaining was performed with Hematoxylin. The immunohistochemical staining results were observed under an optical microscope, and hFIX-positive cells were recorded.

[0159] 3. Experimental Results The results are as follows Figures 7-9 As shown in the figure. Stable expression of hFIX was observed 4 weeks after injection.

[0160] Example 5: In vivo experiments to verify the expression of eGFP after insertion into the last exon of the transferrin gene. The experimental method is the same as in Example 1, except that gRNA1 or gRNA2 in Example 1 is replaced with one of gRNA4-gRNA8. A schematic diagram of the cleavage sites corresponding to gRNA4-gRNA8 is shown below. Figure 10 As shown.

[0161] in Figure 11 The "control" in this context refers to an experimental control that only contains the template and no Cas protein or gRNA.

[0162] The results are as follows Figure 11 As shown, the results indicate that introducing Cas9 cleavage (corresponding gRNAs) into the sequences surrounding the Exon17 sequence of Transferrin effectively improves the insertion and editing efficiency of eGFP (compared to the control group without gRNA), with the greatest benefit observed in cleavage within Exon17. Subsequent experiments used Exon17 cleavage as the test region. Figure 11 The DNA endonuclease is SaCas9.

[0163] Example 6: In vivo experiments to verify the expression of eGFP inserted into the last exon of the transferrin gene. 1. Experimental Methods The method is the same as in Example 3, except that gRNA1 or gRNA2 is replaced with gRNA6, and the DNA endonuclease is SaCas9. This corresponds to the mice being observed continuously for 4 weeks, after which liver tissue was collected for Western blot analysis. The Western blot analysis steps are as follows: (1) Tissue protein extraction process: According to the instructions of Beyotime-RIPA lysis buffer (strong)-tissue sample, 20 mg of frozen mouse liver tissue was lysed with 200 μL of lysis buffer and the supernatant was obtained. The protein concentration range was 31 mg / mL to 38 mg / mL when NanoDrop (Thermo) was subsequently detected.

[0164] (2) SDS-PAGE process: Protein sample thermal denaturation treatment: Tissue sample: 2 x loading buffer, self-prepared. Electrophoresis parameters: 80V electrophoresis to separating gel, 120V electrophoresis to end, the whole process takes about 2.5h.

[0165] (3) Protein transfer process: Conditions are controlled as follows: Low-temperature transfer under ice-water bath conditions: The transfer tank is completely immersed in water containing ice packs to maintain a low temperature (excessive temperature will cause the current to increase and the gel to burn). Transfer parameters: stabilizing voltage 80V, 1.5h.

[0166] (4) Antibody incubation process: The blocking solution was 5% BSA (prepared with TBST), and the membrane was incubated at room temperature with shaking for 1.5 h. (2) Antibody dilution: The antibody was diluted with the blocking solution according to the recommended dilution factor or made as necessary. Primary antibody incubation: overnight incubation. Washing: TBST washing, 5 min / time, 4 times. Secondary antibody incubation: 1.5 h-2 h at room temperature. Washing: TBST washing, 5 min / time, 4-5 times. Imaging: Odyssey CLx infrared fluorescence scanning imaging system was used.

[0167] 2. Experimental Results The results are as follows Figure 12 As shown, no eGFP signal was detected at 27 kDa in the PBS-treated groups (1–3); only weak eGFP protein expression was detected in the Cas9 / gRNA-free groups (4–6), while a clear and significantly enhanced eGFP band appeared at the 27 kDa position in the Cas9 / gRNA3-treated groups (7–9). These results indicate successful transcription and translation of the eGFP gene. Specific cleavage at the Transferrin Exon17 site significantly increases gene insertion efficiency and successfully completes eGFP protein expression. The Actin (42 kDa) expression level was basically consistent in all lanes, indicating uniform loading and transfer.

[0168] The embodiments described in this invention are merely illustrative examples and are not intended to limit the implementation of this invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of this invention should be considered equivalent substitutions and should fall within the protection scope of this invention.

Claims

1. A composition or system, characterized in that, The composition or system comprises: DNA endonuclease or nucleic acid encoding said DNA endonuclease, single-molecule RNA targeting DNA or DNA polynucleotide encoding said single-molecule RNA targeting DNA; and A donor template containing a nucleic acid sequence encoding a foreign protein or a variant thereof or a functional derivative thereof; The single-molecule targeting DNA RNA is an RNA that targets the Transferrin gene.

2. The composition or system according to claim 1, characterized in that, The single-molecule targeting DNA RNA is an RNA that targets the exons of the Transferrin gene; Preferably, the single-molecule DNA-targeting RNA is an RNA that targets exon 17 of the Transferrin gene; Preferably, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs; Preferably, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs; Preferably, the DNA endonuclease is selected from SpCas9 or SaCas9; Preferably, the exogenous protein includes eGFP protein or FIX protein; Preferably, the variant is FIX-padua; Preferably, the sequence of SpCas9 is as shown in SEQ ID NO:1; Preferably, the sequence of SaCas9 is as shown in SEQ ID NO:

2.

3. The composition or system according to claim 1, characterized in that, The single-molecule targeting DNA RNA is sgRNA; Preferably, the composition or system is for inserting a foreign gene or a variant thereof or a functional derivative thereof into exon 17 of the Transferrin gene; Preferably, the exogenous gene includes the eGFP gene or the FIX gene; Preferably, the variant is the FIX-padua gene; Preferably, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22; Preferably, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus; Preferably, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus; Preferably, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10; Preferably, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11; Preferably, the composition or system is used to treat hemophilia; Preferably, the hemophilia is selected from hemophilia type B.

4. A reagent kit, characterized in that, include: (a) A DNA endonuclease or a nucleic acid encoding the DNA endonuclease; (b) A single-molecule targeting DNA RNA or a DNA polynucleotide encoding said single-molecule targeting DNA RNA, wherein said single-molecule targeting DNA RNA is RNA targeting the Transferrin gene; and (c) A donor template containing a nucleic acid sequence encoding a foreign protein or a variant thereof or a functional derivative thereof; Preferably, the single-molecule DNA-targeting RNA is an RNA that targets the exons of the Transferrin gene; Preferably, the single-molecule DNA-targeting RNA is an RNA that targets exon 17 of the Transferrin gene; Preferably, (a), (b), and (c) are in the same or separate containers; Preferably, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs; Preferably, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs; Preferably, the DNA endonuclease is selected from SpCas9 or SaCas9; Preferably, the exogenous protein includes eGFP protein or FIX protein; Preferably, the variant is FIX-padua; Preferably, the sequence of SpCas9 is as shown in SEQ ID NO:1; Preferably, the sequence of SaCas9 is as shown in SEQ ID NO:2; Preferably, the single-molecule targeting DNA RNA is sgRNA; Preferably, the kit is a kit for inserting a foreign gene or its variant or functional derivative into the position of exon 17 of the Transferrin gene; Preferably, the exogenous gene includes the eGFP gene or the FIX gene; Preferably, the variant is the FIX-padua gene; Preferably, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22; Preferably, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus; Preferably, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus; Preferably, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10; Preferably, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11; Preferably, the composition or system is used to treat hemophilia; Preferably, the hemophilia is selected from hemophilia type B.

5. A recombinant carrier composition, characterized in that, include: (1) Endonuclease vector: which includes a DNA endonuclease or a vector encoding the DNA endonuclease; (2) A vector for single-molecule RNA targeting DNA: comprising single-molecule RNA targeting DNA or a DNA polynucleotide encoding said single-molecule RNA targeting DNA; and (3) Donor template-related vectors: These include vectors that encode nucleic acid sequences of exogenous proteins or their variants or functional derivatives; Preferably, the carrier is a plasmid; Preferably, the vector is a virus; Preferably, the virus is an adeno-associated virus; Preferably, the viral vector is a lentiviral expression vector, a retroviral expression vector, an adenovirus expression vector, or an adeno-associated virus expression vector; Preferably, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs; Preferably, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs; Preferably, the DNA endonuclease is selected from SpCas9 or SaCas9; Preferably, the exogenous protein includes eGFP protein or FIX protein; Preferably, the variant is FIX-padua; Preferably, the sequence of SpCas9 is as shown in SEQ ID NO:1; Preferably, the sequence of SaCas9 is as shown in SEQ ID NO:2; Preferably, the single-molecule targeting DNA RNA is sgRNA; Preferably, the recombinant vector composition is used to insert a foreign gene or a variant thereof or a functional derivative thereof into the Transferrin gene; Preferably, the recombinant vector composition is used to insert a foreign gene or a variant thereof or a functional derivative thereof into the exon of the Transferrin gene; Preferably, the recombinant vector composition is a recombinant vector composition for inserting a foreign gene or a variant thereof or a functional derivative thereof into the position of exon 17 of the Transferrin gene; Preferably, the exogenous gene includes the eGFP gene or the FIX gene; Preferably, the variant is the FIX-padua gene; Preferably, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22; Preferably, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus; Preferably, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus; Preferably, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10; Preferably, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:11; Preferably, the composition or system is used to treat hemophilia; Preferably, the hemophilia is selected from hemophilia type B.

6. A recombinant cell, characterized in that, Includes the composition or system according to any one of claims 1-3 or the recombinant viral vector composition according to claim 6; Preferably, the recombinant cells comprise cells that do not express or express low levels of the FIX gene.

7. A pharmaceutical composition for treating a disease, characterized in that, Includes the composition or system according to any one of claims 1-3, the recombinant vector composition according to claim 6, or the recombinant cells according to claim 7; Preferably, the disease includes hemophilia.

8. The use of the composition or system according to any one of claims 1-3, the kit according to any one of claims 4-5, the recombinant vector composition according to claim 6, the recombinant cell according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a disease drug; Preferably, the disease includes hemophilia; Preferably, the hemophilia is hemophilia B.

9. A method for modifying target DNA, characterized in that, This includes contacting the target DNA with a complex, said complex comprising: (a) A DNA endonuclease or a nucleic acid encoding the DNA endonuclease, (b) A single-molecule RNA targeting DNA or a DNA polynucleotide encoding the single-molecule RNA targeting DNA, and (c) A donor template containing a nucleic acid sequence encoding a foreign protein or a variant thereof or a functional derivative thereof; The contact is either outside the body or inside isolated cells; The target DNA is the Transferrin gene; The modification involves bringing the complex into contact with the Transferrin gene, thereby allowing the foreign gene or its variant or functional derivative to be inserted into the Transferrin gene, thus enabling the expression of the foreign protein or its functional derivative simultaneously with the expression of Transferrin. Preferably, the single-molecule DNA-targeting RNA is an RNA that targets the exons of the Transferrin gene; Preferably, the single-molecule DNA-targeting RNA is an RNA that targets exon 17 of the Transferrin gene; Preferably, the DNA endonuclease includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, TALENs, or ZFNs; Preferably, the DNA endonuclease is selected from SpCas9, SaCas9, or TALENs; Preferably, the DNA endonuclease is selected from SpCas9 or SaCas9; Preferably, the exogenous protein includes eGFP protein or FIX protein; Preferably, the variant is FIX-padua; Preferably, the sequence of SpCas9 is as shown in SEQ ID NO:1; Preferably, the sequence of SaCas9 is as shown in SEQ ID NO:2; Preferably, the single-molecule targeting DNA RNA is sgRNA; Preferably, the sgRNA is selected from RNAs complementary to sequences shown in any of SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22; Preferably, the 5' end of the donor template contains a 5' homologous arm that is homologous to the 5' target sequence at the Transferrin genomic locus; Preferably, the 3' end of the donor template contains a 5' homologous arm that is homologous to the 3' target sequence at the Transferrin genomic locus; Preferably, the sequence of the 5' homologous arm is as shown in SEQ ID NO:8 or SEQ ID NO:10; Preferably, the sequence of the 3' homologous arm is as shown in SEQ ID NO:9 or SEQ ID NO:

11.

10. An sgRNA, characterized in that, It is selected from RNAs complementary to any of the sequences shown in SEQ ID NO:5 to SEQ ID NO:7 and SEQ ID NO:18 to SEQ ID NO:22.

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