CN110885799A

CN110885799A - Gene editing method based on adenovirus

Info

Publication number: CN110885799A
Application number: CN201911053013.4A
Authority: CN
Inventors: 史其萍; 贺小宏; 任江涛; 王延宾; 韩露
Original assignee: Nanjing North Heng Biological Technology Co Ltd
Current assignee: Nanjing North Heng Biological Technology Co Ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-03-17
Also published as: WO2021082784A1

Abstract

The present invention provides a recombinant adenovirus comprising: the target expression cassette comprises a target expression cassette, a first homology arm positioned at the 5 'end of the target expression cassette, a second homology arm positioned at the 3' end of the target expression cassette, and one or two sgRNA target sequences, wherein the sgRNA target sequences are positioned at the 5 'end of the first homology arm or the 3' end of the second homology arm, or positioned at the 5 'end of the first homology arm and the 3' end of the second homology arm respectively; the invention also provides a gene editing method based on the recombinant adenovirus, a method for preparing a gene editing animal, a method for preparing an engineered T cell, and a system, a composition and a kit containing the recombinant adenovirus.

Description

Gene editing method based on adenovirus

Technical Field

The present invention relates to the field of gene editing. In particular, the present invention relates to an adenovirus-based gene editing method.

Background

Chimeric antigen receptor T Cells (CART) are one of the most promising tumor immunotherapy at present, and the basic principle is mainly to extract the T cells of a patient, express specific chimeric antigen receptors through gene and cell engineering means, enable the chimeric antigen receptors to recognize and combine with tumor cell surface antigens, and thus play a role in targeted killing of tumor cells. CAR-T cell therapy is currently approved by the FDA in the united states for the treatment of Acute Lymphoblastic Leukemia (ALL), and relapsed or refractory large B-cell lymphoma (LBCL) adult patients, including diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), high grade B-cell lymphoma (HGBL), and Transformed Follicular Lymphoma (TFL).

At present, CART production is generally performed using transposon DNA vectors (e.g., plasmids) in a lentiviral, retroviral, adeno-associated viral or non-viral format. However, lentiviruses or retroviruses randomly insert into the genome after entering cells, which may destroy other genes in the cells, thereby causing cellular abnormalities, and may even allow the cells to transform into tumor cells, thereby causing tumors. Therefore, the cellular immunotherapy using these viral technologies to prepare CART has the risk of tumor initiation. The possibility of random insertion is reduced by non-viral transposon DNA vectors, but the risk of random insertion remains, and transduction of foreign DNA into T cells in this way is cytotoxic and the clinical efficacy is unsatisfactory. The CART prepared by adopting the adeno-associated virus can be inserted into a target gene at a fixed point to solve the problem of random insertion, but the production and purification process is complex and cannot be well popularized clinically.

Therefore, there is a need for a method and system for performing site-directed gene editing in a cell that overcomes the deficiencies of the above-described techniques.

Disclosure of Invention

The invention aims to provide an efficient adenovirus-based targeted gene editing method and system, which can solve the problem of random insertion and are suitable for industrial scale production and clinical application. It is also an object of the invention to provide uses of the gene editing methods, for example in the treatment of disease.

Accordingly, in a first aspect, the present invention provides a recombinant adenovirus comprising: the expression cassette comprises a target expression cassette, a first homology arm positioned at the 5 'end of the target expression cassette, a second homology arm positioned at the 3' end of the target expression cassette, and one or two sgRNA target sequences, wherein the sgRNA target sequences are positioned at the 5 'end of the first homology arm or the 3' end of the second homology arm, or positioned at the 5 'end of the first homology arm and the 3' end of the second homology arm respectively.

In one embodiment, the expression cassette of interest comprises a sequence for engineering the genome of a cell, such as a coding sequence for a Chimeric Antigen Receptor (CAR).

In one embodiment, the first homology arm and the second homology arm are complementary to the 300-. In a preferred embodiment, the first homology arm and the second homology arm are complementary to sequences of any length between 400 and 2500bp, such as 400bp, 600bp, 800bp, 1000bp, 1200bp, 1400bp, 1600bp, 1800bp, 2000bp, 2200bp, 2400bp or 2500bp, or 400 and 2500bp, respectively, at both ends of the target gene sequence of the cellular genome. The first and second homology arms may be the same or different in length.

In one embodiment, a recombinant adenovirus of the invention includes a sgRNA target sequence located 5 'of the first homology arm or 3' of the second homology arm. In another embodiment, a recombinant adenovirus of the invention comprises two sgRNA target sequences located at the 5 'end of the first homology arm and the 3' end of the second homology arm, respectively, and the two sgRNA target sequences may be the same or different.

In one embodiment, the recombinant adenovirus of the invention further comprises a promoter, an Inverted Terminal Repeat (ITR), and/or a packaging signal. Examples of recombinant adenoviruses include, but are not limited to, Ad5, Ad5F35, Ad35, Ad55, Ad2, Ad5F11, pAdBM5, pADCMV5, and the like.

In one embodiment, the expression cassette of interest, the first homology arm, the second homology arm, and the sgRNA target sequence in the recombinant adenovirus are operably linked to each other.

In a second aspect, the present invention provides an adenovirus-based gene editing method comprising:

(1) providing a recombinant adenovirus of the invention;

(2) and taking the recombinant adenovirus as a donor, and carrying out gene editing on a cell genome through a CRISPR/Cas system in the presence of sgRNA of a target gene sequence of the target cell genome and sgRNA of a target sgRNA target sequence.

In one embodiment, the cell is a mammalian cell, preferably a human cell. In another embodiment, the cells include, but are not limited to, stem cells, such as embryonic stem cells, pluripotent stem cells, adult stem cells, and the like, or somatic cells. In another embodiment, examples of the cells include, but are not limited to, hematopoietic stem cells, T cells, B cells, dendritic cells, macrophages, natural killer cells (NK cells), monocytes, embryonic stem cells, induced pluripotent stem cells, and the like.

In one embodiment, the sgRNA targeting the target gene sequence of the genome of the cell, the sgRNA targeting the sgRNA target sequence, may be the same or different. In one embodiment, the sgrnas (including the sgRNA targeting a gene sequence of interest of a cellular genome and the one or two sgrnas targeting the one or two sgRNA target sequences) are provided as RNAs, or as polynucleotides encoding the sgrnas. Where provided in the form of a polynucleotide encoding a sgRNA, the polynucleotide can be present on one or more expression vectors.

In one embodiment, the Cas protease in the CRISPR/Cas system is Cas9 or Cpf 1. The Cas protease is provided in the form of mRNA encoding it, or in the form of a polynucleotide encoding it, or in the form of a protein. When provided in the form of an encoding polynucleotide, it may be present in the same or a different expression vector as the expression vector providing the sgRNA.

In one embodiment, a recombinant adenovirus, sgRNA or an expression vector providing the sgRNA, mRNA encoding the Cas protease or an expression vector providing the Cas protease are delivered together or separately into the cell.

In one embodiment, gene editing includes, but is not limited to, insertion, deletion, or substitution of gene segments, mutation of one or more bases, genetic modification, and the like.

In one embodiment, the length of the gene fragment to which the insertion, deletion or substitution is made may be 1bp to 30kb, for example 1bp (e.g.point mutation), 100bp, 1kb, 5kb, 10kb, 15kb, 20kb, 25kb or 30kb, or any length between 1bp and 30 kb.

In one embodiment, the gene editing methods of the invention are not aimed at diagnosing or treating a disease.

In a third aspect, the invention provides a system, composition or kit for gene editing comprising a recombinant adenovirus of the invention.

In a preferred embodiment, the system, composition, or kit further comprises one or two sgrnas or polynucleotides encoding thereof that target one or two sgRNA target sequences in the recombinant adenovirus.

In another preferred embodiment, the system or kit further comprises mRNA or polynucleotide encoding a Cas protease. In a preferred embodiment, the Cas protease is Cas9 or Cpf 1.

In the present invention, when the sgRNA or Cas protease is provided in the form of a polynucleotide, the polynucleotide may be present in one or more expression vectors.

In a fourth aspect, the present invention provides a method of producing a gene-edited animal, comprising performing gene editing on a fertilized egg of the animal using the gene editing method of the present invention, and allowing the fertilized egg to develop, thereby obtaining an animal in which targeted gene editing has occurred.

In a preferred embodiment, the animal is a mouse, rat or zebrafish.

In a fifth aspect, the invention provides a method of making an engineered T cell, comprising:

(1) providing a recombinant adenovirus of the invention;

(2) delivering the recombinant adenovirus, the sgRNA or its encoding polynucleotide targeting the target gene sequence of the T cell genome, the sgRNA or its encoding polynucleotide targeting the sgRNA target sequence, and the mRNA or polynucleotide encoding the Cas protease into a T cell, thereby obtaining an engineered T cell.

In one embodiment, the Cas protease is Cas9 or Cpf 1.

In one embodiment, the T cells are derived from Peripheral Blood Mononuclear Cells (PBMCs) or umbilical cord blood. Preferably, the T cells include, but are not limited to, inflammatory, cytotoxic, regulatory or helper T cells, more preferably CD4+ and/or CD8+ T cells.

In one embodiment, a polynucleotide encoding a sgRNA that targets a T cell genome target gene sequence, a polynucleotide encoding a sgRNA that targets a sgRNA target sequence, and a polynucleotide encoding a Cas protease are present on one or more expression vectors.

The present invention also provides an engineered T cell obtained by the above preparation method and a composition comprising the engineered T cell.

In one embodiment, the engineered T cell is a TCR T cell or a CART cell. In one embodiment, the CART cell is a universal CART cell. In another embodiment, the engineered T cells can be used to treat a disease, such as cancer, an infectious disease, or an autoimmune disease.

Detailed Description

Definition of

As used herein, the term "gene editing" refers to a technique for making precise modifications to a gene of an organism. Specific genes in a cell can be mutated, knocked in, deleted, etc. by gene editing techniques, thereby changing the genetic characteristics of an organism. Current gene editing techniques use methods to cause DNA damage at specific locations in a gene, thereby triggering a DNA damage repair mechanism within the cell.

The current DNA damage repair mechanisms mainly include two pathways, Non-Homologous end joining (NHEJ) and Homologous Recombination (HR). In the non-homologous end joining approach, the cell partially cleaves the DNA at the break, creating a sticky end, and joins the two ends. Although this approach is rapid and efficient, it often results in deletion or insertion of some bases, making gene editing by this approach less accurate.

In the homologous recombination pathway, another intact DNA having high homology with the DNA damage site is introduced into the cell, and the damage site is repaired by using the homologous DNA as a template. Modification of the cellular genome can be achieved if other genes, elements or point mutations are introduced into the homologous genes. Therefore, the repair of genomic DNA by homologous recombination has higher controllability, can completely generate expected genomic changes, and can directionally introduce specific exogenous genes at proper positions. For example, a specific gene can be inserted into a relatively stable genomic position for expression, which is particularly important for obtaining a highly expressed recombinant protein cell line. However, during gene editing, for example by CRISPR systems, the efficiency of the NHEJ produced is often much higher than HR, which is undesirable for targeted precise gene repair.

As used herein, the term "adenovirus" has the ordinary meaning as understood by those skilled in the art and refers to a macromolecular double-stranded non-enveloped DNA virus. Following entry into the cell by receptor-mediated endocytosis, the adenovirus genome is transferred into the nucleus, but remains extrachromosomal and does not integrate into the genome of the host cell. The adenoviral genome comprises two Inverted Terminal Repeats (ITRs), a viral packaging signal located inside the ITRs, 4 transcriptional units (E1-E4) expressed in early phase associated with adenoviral replication and 1 transcriptional unit encoding a structural protein expressed in late phase. More than 100 serotypes have been discovered, of which there are 52 human adenoviruses, designated Ad1-Ad52, respectively. Examples of adenoviruses that can be used in the present invention include, but are not limited to, Ad2, Ad5, Ad35, and the like. Other chimeric adenoviruses, such as Ad5F35 (modified receptor-binding fiber regions based on conventional Ad5 type adenovirus), Ad5F11, Ad55, Ad5F11, pAdBM5, and pADCMV5, are also useful in the invention.

As used herein, the term "recombinant adenovirus" refers to an adenovirus produced by genetic engineering, wherein the adenovirus genome is engineered so that it is suitable for expression of a foreign gene. In general, recombinant adenoviruses have deletions of some or all of the native genes. In the present invention, the recombinant adenovirus E2 region encodes a DNA binding protein with a mutation in the gene that reduces the cellular immune response caused by expression of the viral protein. For example, the recombinant adenovirus may lack one or more of the E1, E2, E3, E4 genes. Alternatively, the adenovirus may delete all or most of the adenovirus genes, leaving only the ITRs and the packaging signal sequences.

As used herein, the term "expression cassette of interest" refers to a sequence for gene replacement with a gene sequence of interest of a genome of a cell. After the gene editing is completed, part or all of the target expression cassette replaces the target gene sequence, thereby integrating into the genome of the cell. Thus, the expression cassette of interest comprises sequences for engineering the genome of the cell, such as the foreign gene to be inserted, the coding sequence of a Chimeric Antigen Receptor (CAR), and the like.

As used herein, the term "homology arm" refers to a sequence that is homologous complementary to a target gene sequence. The length of the homology arm can be 400-2500bp, such as any length between 400bp, 600bp, 800bp, 1000bp, 1200bp, 1400bp, 1600bp, 1800bp, 2000bp, 2200bp, 2400bp or 2500bp, or 400-2500 bp.

As used herein, the term "sgRNA" refers to a single guide rna (single guide rna) that comprises crRNA and tracrRNA. sgrnas are designed based on specific target sites on the target gene sequence, with a sequence sufficient to act synergistically with Cas9 or Cpf1 endonucleases to guide the occurrence of DNA double strand breaks at the endonuclease-mediated target sites.

As used herein, the term "sgRNA target sequence" refers to a sequence that is recognized by and binds to a sgRNA.

As used herein, the term "operably linked" refers to a functional spatial arrangement between two or more polynucleotide fragments. For example, a promoter is operably linked to a DNA sequence if it stimulates or regulates the transcription of the DNA sequence in an appropriate host cell or other expression system. Two or more polynucleotide fragments may be joined by a linker to achieve an operably linked relationship.

As used herein, the term "CRISPR" refers to clustered regularly interspaced short palindromic repeats. CRISPRs were originally described as prokaryotic DNA fragments containing short repetitive base sequences. In a palindromic repeat sequence, the sequence of nucleotides is identical in both directions. Each repeat is followed by a short segment of spacer DNA from a previous exposure to foreign DNA (e.g., a virus or plasmid). CRISPR sites typically consist of: a cluster of CRISPR-associated (Cas) genes and a characteristic CRISPR array-a series of repeated sequences (direct repeats) separated by a variable sequence (spacer) that corresponds to a sequence in a foreign genomic element (protospacer). When the Cas gene is translated into a protein, most CRISPR arrays are first transcribed into a single RNA, which is then processed into shorter CRISPR RNA (crRNA) that directs the nucleolytic activity of certain Cas enzymes to degrade the target nucleic acid.

As used herein, the term "CRISPR/Cas system" refers to a prokaryotic immune system that confers resistance to foreign genomic elements (such as those present in plasmids and phages) that provides one form of acquired immunity. Typically, the CRISPR/Cas system comprises at least one Cas endonuclease and a guide RNA. The RNA carrying the spacer sequence helps the Cas (CRISPR-associated) protein recognize and cleave the exogenous DNA. Thus, when the Cas protein is Cas9, the system is referred to as CRISPR/Cas9 system; when the Cas protein is Cpf1, the system is referred to as a CRISPR/Cpf1 system.

As used herein, the term "Cas 9" refers to a Cas protein found in streptococcus pyogenes. Cas9 endonuclease is a four-component system comprising two small RNA molecules called CRISPR RNA (crRNA) and transactivation CRISPR RNA (tracrRNA).

As used herein, the term "Cpf 1" refers to an RNA-guided DNA endonuclease belonging to the class II subtype V-a CRISPR system, obtained from prevotella and francisella 1. The Cpf1 endonuclease includes a conserved RuvC nuclease domain, known to hydrolyze single-stranded dna (ssdna) and a second catalytic domain, which is responsible for the independent processing of self-crRNA. It was reported that crRNA maturation by Cpf1 did not require the assistance of tracrRNA.

In one embodiment, the Cas enzymes of the present invention also include functional variants thereof. By "functional variant" is meant a variant that is biologically active, i.e., it comprises one or more functional properties of the parent protein. In the context of the present invention, a "functional variant" has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of a parent protein. A functional variant may be obtained by mutation of the wild-type parent protein, resulting in the addition, deletion or substitution of one or more amino acids. Methods of mutagenesis are known in the art, such as random mutagenesis or site-directed mutagenesis. For example, dlcpcf 1 is a functional variant of LbCpf1, which contains a D832A mutation, resulting in loss of catalytic activity of its DNA endonuclease. "functional variants" also include chimeric proteins comprising a first fragment from a first protein and a second fragment from a second protein, wherein the first and second proteins are different.

As used herein, the term "CAR" or "chimeric antigen receptor" refers to an engineered receptor that specifically transplants any antigen onto immune effector cells (T cells). Typically, these receptors are used to graft the specificity of monoclonal antibodies onto T cells. These receptors are called chimeras because they are composed of portions of different origin.

As used herein, the term "CAR T cell" or "chimeric antigen receptor T cell" refers to an engineered T cell having a chimeric antigen receptor with a predefined specificity for a selected target. Upon encountering a target cell, e.g., a cancer cell, the CAR T cell destroys the cancer cell by, for example, the following mechanisms: the general stimulation of cell proliferation, increasing the degree to which cells are toxic to other living cells (i.e., cytotoxicity), and increasing the production of factors secreted by cells in the immune system, which have an effect on other cells within the organism.

As used herein, the terms "universal CART cell" or "UCART cell" are used interchangeably and refer to the generation of graft-versus-host disease (GVHD) and graft rejection in Human Lymphocyte Antigen (HLA) non-matched recipients by gene editing of healthy donor T cells to prevent their expression of endogenous T Cell Receptors (TCRs) and to obtain clinically useful CAR-T cells in GMP laboratories. Universal CART cells are an allogeneic, ready-to-use T cell product.

Recombinant adenovirus

The present invention provides a recombinant adenovirus comprising: the expression cassette comprises a target expression cassette, a first homology arm positioned at the 5 'end of the target expression cassette, a second homology arm positioned at the 3' end of the target expression cassette, and one or two sgRNA target sequences, wherein the sgRNA target sequences are positioned at the 5 'end of the first homology arm or the 3' end of the second homology arm, or positioned at the 5 'end of the first homology arm and the 3' end of the second homology arm, respectively.

In one embodiment, a recombinant adenovirus of the invention includes a sgRNA target sequence located 5 'of the first homology arm or 3' of the second homology arm. In another embodiment, a recombinant adenovirus of the invention comprises two sgRNA target sequences located at the 5 'end of the first homology arm and the 3' end of the second homology arm, respectively, and the two sgRNA target sequences may be the same or different. For example, two sgRNA target sequences may be designed to be the same sequence, and only one sgRNA is required to form gaps at the 5 'end of the first homology arm and the 3' end of the second homology arm, respectively, so that a DNA fragment consisting of the target expression cassette and the two homology arms is inserted into the cell genome through complementary pairing between the sequences at the two ends of the first homology arm, the second homology arm, and the target gene sequence, thereby replacing the target gene sequence.

In one embodiment, the expression cassette of interest comprises sequences for engineering the genome of a cell. For example, the expression cassette of interest may comprise a deletion of a gene fragment or insertion of a foreign gene, or a mutation of one or more bases, as compared to the wild-type gene sequence, such that deletion of a gene fragment, insertion of a foreign gene, point mutation, genetic modification, or gene sequence replacement occurs directionally in the genome of the cell when the expression cassette of interest is integrated into the genome of the cell. In a preferred embodiment, the expression cassette of interest comprises a coding sequence, such as a reporter gene, a structural gene, a functional gene, or a Chimeric Antigen Receptor (CAR).

In one embodiment, the recombinant adenovirus of the invention further comprises a promoter, Inverted Terminal Repeat (ITR), and/or packaging signal sequence. Examples of recombinant adenoviruses include, but are not limited to, pAd5, pAd5F35, pAd35, Ad55, Ad2, Ad5F11, pAdBM5, pADCMV5, and the like.

In one embodiment, the individual elements in the recombinant adenovirus, such as the expression cassette of interest, the first homology arm, the second homology arm, the sgRNA target sequence, the optional promoter, the ITRs and the packaging signal sequence, are operably linked to each other.

The principles and methods for designing and synthesizing the various elements in a recombinant adenovirus, including but not limited to the expression cassette of interest, first homology arm, second homology arm, sgRNA target sequence, and optional promoter, are well known to those skilled in the art. Depending on the cell or species in which gene editing is desired, one skilled in the art can design elements with different sequences to form adenoviruses with different sequences to achieve targeted gene editing.

Gene editing method and application thereof

The invention provides an adenovirus-based gene editing method, which comprises the following steps:

(1) a recombinant adenovirus is provided, comprising: the target expression cassette comprises a target expression cassette, a first homology arm positioned at the 5 'end of the target expression cassette, a second homology arm positioned at the 3' end of the target expression cassette, and one or two sgRNA target sequences, wherein the sgRNA target sequences are positioned at the 5 'end of the first homology arm or the 3' end of the second homology arm, or positioned at the 5 'end of the first homology arm and the 3' end of the second homology arm respectively;

(2) and taking the recombinant adenovirus as a donor, and carrying out gene editing on a cell genome through a CRISPR/Cas system in the presence of sgRNA of a target gene sequence of the targeted cell genome and sgRNA of a target sgRNA target sequence.

The method of the invention can be carried out in essentially any eukaryotic cell. Thus, in one embodiment, the cell is an animal cell, a plant cell or a microbial cell. In a preferred embodiment, the cell is a mammalian (e.g., human, non-human primate, mouse, rat, rabbit, pig, sheep, horse, cow, etc.) cell, preferably a human cell. In another embodiment, the cells include, but are not limited to, stem cells, such as embryonic stem cells, pluripotent stem cells, adult stem cells, and the like, or somatic cells. In another embodiment, examples of the cells include, but are not limited to, hematopoietic stem cells, T cells, B cells, dendritic cells, macrophages, natural killer cells (NK cells), monocytes, embryonic stem cells, induced pluripotent stem cells, and the like.

In one embodiment, the sgRNA targeting the genomic target gene sequence of the cell and the sgRNA targeting the sgRNA target sequence may be the same or different. For example, the sgRNA target sequence in the genome of the cell and the sgRNA target sequence in the adenovirus can be designed to be identical or complementary sequences, so that the sgRNA can be cleaved with the same sgRNA, thereby making the operation easier.

In one embodiment, the sgrnas (including the sgRNA targeting a gene sequence of interest of a cellular genome and the one or two sgrnas targeting the one or two sgRNA target sequences) are provided as RNAs, or as polynucleotides encoding the sgrnas. Where provided in the form of a polynucleotide encoding a sgRNA, the polynucleotide can be present on one or more expression vectors.

In one embodiment, the Cas enzyme in the CRISPR/Cas system may be a wild-type protease or a functional variant, e.g., a functional variant or a chimeric protein, that retains the endonuclease activity of the wild-type protease. Functional variants can be obtained by any known method (e.g., directed or random mutagenesis or DNA recombination) by those skilled in the art. The effect of the obtained functional variants can be verified by well-known methods, such as DNA cleavage analysis.

In one embodiment, the Cas enzyme is Cas9 or Cpf 1. Cas9 or CPf1 is provided in the form of mRNA encoding it, or in the form of a polynucleotide encoding it. When provided in the form of an encoding polynucleotide, it may be present in the same or a different expression vector as the expression vector providing the sgRNA.

In one embodiment, a recombinant adenovirus, sgRNA or an expression vector providing sgRNA, Cas9 or Cpf1mRNA or an expression vector providing Cas9 or Cpf1 protein are delivered together or separately into a cell. Delivery can be by any method known to those skilled in the art, such as by electroporation, particle gun methods, microinjection, liposomes, calcium phosphate methods, nanoparticles, and the like.

In one embodiment, gene editing includes, but is not limited to, insertion, deletion, or substitution of gene segments, mutation of one or more bases, genetic modification, and the like. Specifically, the purpose of gene editing is achieved by designing a specific target expression cassette, for example, the target expression cassette is designed to further include a gene fragment to be inserted or delete a certain length of the gene fragment as compared with the target gene sequence of the cellular genome, so that after gene editing by the method of the present invention, the target gene sequence of the cellular genome is replaced by the target expression cassette, thereby allowing the cellular genome to be directionally subjected to insertion or deletion of the gene fragment. The expression cassette of interest may also be designed to contain a mutation of one or more bases compared to the target gene sequence of the genome of the cell, such that the mutation of the one or more bases occurs directionally in the genome of the cell after the expression cassette of interest replaces the target gene sequence.

The present invention also provides a method for producing a gene-edited animal, comprising performing gene editing on a fertilized egg of an animal using the gene editing method of the present invention, and allowing the fertilized egg to develop, thereby obtaining an animal in which targeted gene editing has occurred. Such genetically edited animals can be used as animal models for laboratory or clinical studies of disease development mechanisms, tumor progression mechanisms, potential therapeutic approaches or for assessing the efficacy of treatment, etc. For example, a gene-edited mouse, rat, zebrafish, or the like can be obtained by the gene editing method of the present invention.

The editing methods of the invention can also be used to prepare engineered T cells to express modified TCRs (i.e., TCR therapy) or Chimeric Antigen Receptors (CARs) with enhanced antigen specificity.

Genetically modified TCR therapy is based on altering the specificity of T cells through the expression of specific TCR α and β chains that mediate the antigen recognition process tumor-specific TCR α and β chains were identified, isolated and cloned into transduction vectors, and the transduction of T cells produced tumor antigen-specific T cells.

Chimeric Antigen Receptors (CARs) combine antibody-like recognition and T cell activation functions. They consist of an antigen binding region, usually derived from an antibody, a transmembrane domain that anchors the CAR to a T cell, and one or more intracellular signaling domains that induce persistence, trafficking, and effector functions in transduced T cells. The sequences used to define the CAR antigen targeting motif are typically derived from monoclonal antibodies, although ligands and other receptors may also be used.

Accordingly, the present invention also provides a method of making an engineered T cell comprising:

(1) providing a recombinant adenovirus of the invention;

(2) delivering the recombinant adenovirus, the sgRNA or its encoding polynucleotide targeting the target gene sequence of the T cell genome, the sgRNA or its encoding polynucleotide targeting the sgRNA target sequence, and the mRNA or polynucleotide encoding Cas9 or Cpf1 into a T cell, thereby obtaining an engineered T cell.

In one embodiment, the recombinant adenovirus, the sgRNA or its encoding polynucleotide targeting a gene sequence of interest of the T cell genome, the sgRNA or its encoding polynucleotide targeting the sgRNA target sequence, and the mRNA or polynucleotide encoding Cas9 or Cpf1 can be delivered into the T cell together or separately. Methods of delivery are well known to those skilled in the art.

In one embodiment, a polynucleotide encoding a sgRNA that targets a gene sequence of interest of a T cell genome, a polynucleotide encoding a sgRNA that targets a sgRNA target sequence, and a polynucleotide encoding Cas9 or Cpf1 are present on one or more expression vectors.

In one embodiment, the engineered T cell is a TCR T cell or a CART cell. In a preferred embodiment, the CART cell is a universal CART cell.

The engineered T cells according to the invention have a wide range of uses, for example as active ingredients in pharmaceutical composition products for the treatment or prevention of diseases such as cancer, infections or autoimmune diseases, ideally as "off-the-shelf products.

Disorders that can be treated by engineered T cells include, but are not limited to, cancer, infection, or autoimmune disease. Cancers that can be treated with engineered T cells include, but are not limited to, Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), breast cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, metastatic adenocarcinoma, liver metastases, sarcoma, osteosarcoma, neuroblastoma, melanoma, mesothelioma, glioblastoma, glioma, malignant glioma, hepatocyte, non-small cell lung cancer (NSCLC), ganglioneuroma, brain cancer, renal cancer, and prostate cancer. Infections that can be treated with engineered T cells include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites. Autoimmune diseases that can be treated with engineered T cells include, but are not limited to, type I diabetes, celiac disease, graves 'disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, addison's disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus.

Herein, the tumor antigen is selected from TSHR, CD123, CD171, CS-1, CLL-1, CD, EGFRvIII, GD, BCMA, TnAg, PSMA, ROR, FLT, FAP, TAG, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, mesothelin, IL-l Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-, SSEA-4, CD, folate receptor, ERBB (Her/neu), MUC, EGFR, NCAM, prostasin, PAP, ELF2, Ephrin B, IGF-I receptor, CAIX, LMP, gOOpl, bcr-abl, EphA, tyrosyl, fucol, sLe, GM, TGS, HMGA-7, WM-ATRP, MGRA, MAGE-7-LR, MAGE-7, MAGE, MAG-7, MAGE, MAG-7, MAG-E, MAGE, MAG-E, MAG-E, MAG-E, MAG.

System, composition or kit for gene editing

The invention also provides a system, composition or kit for gene editing comprising a recombinant adenovirus of the invention.

In a preferred embodiment, the system, composition or kit further comprises: one or two sgrnas or polynucleotides encoding same that target one or two sgRNA target sequences in a recombinant adenovirus.

In another preferred embodiment, the system or kit further comprises an mRNA or polynucleotide encoding a Cas protease, such as Cas9 or Cpf1mRNA or a polynucleotide encoding same.

In the present invention, when the sgRNA or Cas9 or Cpf1 is provided in the form of a polynucleotide, the polynucleotide may be present in one or more expression vectors.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: schematic representation of different structures of adenovirus causing recombination; a: the recombinant adenovirus does not comprise sgRNA target sequences. In this case, the first and second homology arms in the adenovirus genome are inserted into the cell genome by complementary pairing with the sequences at both ends of the target gene sequence in the cell genome, thereby replacing the target gene sequence, but the efficiency of such insertion is extremely low because the adenovirus genome sequence is large (about 40 kb); b and C: the recombinant adenovirus comprises two sgRNA target sequences (of the invention) located at the 5 'end of the first homology arm (sgRNA-1 target) and the 3' end of the second homology arm (sgRNA-2 target), respectively. By adding sgRNA targeting a sgRNA target sequence and sgRNA targeting a target gene sequence in a cell genome, a DNA fragment consisting of a first homologous arm, a target expression frame and a second homologous arm can be cut from an adenovirus genome while a double-strand break gap is formed on the cell genome through a CRISPR system, and the DNA fragment can be inserted into a gap (B) in the cell genome through an NHEJ pathway or can directly replace the target gene sequence (C) in the cell genome through an HR pathway; d: the recombinant adenovirus comprises a sgRNA target sequence (the invention) and is positioned at the 5' end (sgRNA-4 target) of the first homologous arm, in this case, the sgRNA added with the target gene sequence in the genome of the target cell can cut the target gene sequence in the genome of the cell to form a gap, the sgRNA added with the target gene sequence of the sgRNA can cut the genome of the recombinant adenovirus to linearize the recombinant adenovirus, and then the first homologous arm and the second homologous arm in the genome of the adenovirus are inserted into the genome of the cell through complementary pairing with the sequences at two ends of the target gene sequence in the genome of the cell, thereby replacing the target gene sequence.

FIG. 2: using adenovirus vector to carry out site-specific gene insertion in 293T cells; 1. transfecting pAd5F35-TRAC-CAR-HDR plasmid and Cas9mRNA and sgRNA simultaneously; 2. transfecting the pAd5F35-TRAC-CAR-HDR plasmid and transfecting Cas9 mRNA; 3. transfecting pAd5F35-sgTRAC-CAR-HDR plasmid and Cas9mRNA and sgRNA simultaneously; 4. transfecting pAd5F35-sgTRAC-CAR-HDR plasmid and transfecting Cas9 mRNA; 5. transfecting Cas9mRNA and sgRNA only; 6. transfection of Cas9mRNA only; the arrows show the target bands.

FIG. 3: HR and NHEJ mediated site-directed gene insertion in a549 cells using adenovirus; i-1 to I-3: pAd5F35-sgTRAC-CAR-HDR adenovirus alone was added, and the MOIs of the adenoviruses in I-1 to I-3 were 5X10, respectively³、2.5X10³And 1X10³(ii) a I-4: complete blank control (Cas 9mRNA and sgRNA untransfected and no adenovirus added); II-1 to II-3: pAd5F35-sgTRAC-CAR-HDR adenovirus was added and transfected with Cas9mRNA, and MOI of adenovirus in II-1 to II-3 was 5X10, respectively³、2.5X10³And 1X10³(ii) a II-4: transfection of Cas9mRNA only; III-1 to III-3: pAd5F35-sgTRAC-CAR-HDR adenovirus is added and Cas9mRNA and sgRNA are transfected at the same time, and MOI of adenovirus in III-1 to III-3 is 5X10³、2.5X10³And 1X10³(ii) a III-4: transfecting Cas9mRNA and sgRNA only; the arrows show the target bands of approximately 1000bp size and 2000bp size.

FIG. 4: HR-mediated site-directed gene insertion in T cells using adenovirus; 1. adding pAd5F35-sgTRAC-CAR-HDR adenovirus and transfecting Cas9mRNA and sgRNA simultaneously; 2. adding pAd5F35-sgTRAC-CAR-HDR adenovirus and transfecting Cas9 mRNA; 3. complete blank control (Cas 9mRNA and sgRNA untransfected and no adenovirus added); the arrows show the target bands.

FIG. 5: the NHEJ-mediated insertion can be avoided by nicking on the host genome and adenovirus respectively; 1. adding pAd5F35-sgTRAC-CAR-HDR adenovirus and transfecting Cas9mRNA and genome-targeted sgRNA (TRAC-sgRNA) simultaneously; 2. adding pAd5F35-sgTRAC-CAR-HDR adenovirus and simultaneously transfecting Cas9mRNA, genome-targeted sgRNA (TRAC-sgRNA) and adenovirus left arm single-cutting sgRNA (sgRNA-4); the arrows show the target bands.

FIG. 6: results of transfection of a549 cells and 293T cells with vector plasmids and adenovirus; MFI: average fluorescence intensity; analysis was performed using Two-way ANOVA and statistical analysis was performed using T test; denotes a P value of less than 0.05, denotes a P value of less than 0.01, reaching a significant level; ns indicates no significant difference.

Detailed Description

Unless otherwise indicated, the specific test procedures in the following examples were carried out according to conventional conditions such as those described in molecular cloning guidelines, third edition, published by scientific publishers, J. SammBruke et al, or according to conditions recommended by the manufacturer.

Example 1

Construction of adenovirus vectors

1. Construction of shuttle vectors

Synthesizing sgTRAC-CAR-HDR comprising, in order: a TRAC sgRNA target sequence (SEQ ID NO: 1), a first homologous arm (SEQ ID NO: 2), a target gene CD19-CD22 CAR (SEQ ID NO: 3), a second homologous arm (SEQ ID NO: 4) and a TRAC sgRNA target sequence (SEQ ID NO: 1). Synthesizing a TRAC-CAR-HDR comprising, in order: a first homology arm (SEQ ID NO: 2), a target gene TRAC (SEQ ID NO: 3) and a second homology arm (SEQ ID NO: 4). sgTRAC-CAR-HDR and TRAC-CAR-HDR were ligated into pSIREN-Shuttle Shuttle vector (Clontech, cat # 631527), respectively, by enzymatic cleavage.

2. Construction of adenovirus vector plasmids

The sgTRAC-CAR-HDR and TRAC-CAR-HDR sequences in the shuttle vector were cloned into pAd5F35 adenovirus vectors using PI-SceI/I-CeuI endonuclease to obtain pAd5F35-sgTRAC-CAR-HDR (FIG. 1B-C) and pAd5F35-TRAC-CAR-HDR (FIG. 1A) vector plasmids.

Example 2

Site-directed gene insertion using adenovirus vector plasmids

293T cells were digested with 0.05% Trypsin-EDTA (1X) (Gibco, cat # 25300-. Then, the Transfection Reagent X-tremeGENE HP DNA Transfection Reagent (Roche, cat # 06366546001) and pAd5F35-sgTRAC-CAR-HDR or pAd5F35-TRAC-CAR-HDR vector plasmid 2:1 (v/w) mixed well, at room temperature for 15min, slowly added to 293T cells. After 1 day, 10. mu.g of sgRNA targeting the TRAC gene (SEQ ID NO: 5) was transfected into 293T cells that had been infected with the vector plasmid, in the same manner as described above for transfection of Cas9 mRNA. 293T cells not infected with vector plasmid were transfected with sgRNA and Cas9mRNA targeting the TRAC gene, or Cas9mRNA alone as controls. After 3 days of transfection, genomic DNA of 293T cells was extracted and gene insertion was verified by PCR amplification. The primers used for PCR amplification were: PCR-F (SEQ ID NO: 7) on genomic DNA and PCR-R (SEQ ID NO: 8) on a vector plasmid.

And (3) performing gel electrophoresis on the PCR amplification product, and displaying that: neither transfection of Cas9mRNA and sgRNA simultaneously, nor transfection of Cas9mRNA alone, the target band could be obtained using the pAd5F35-TRAC-CAR-HDR vector plasmid. Target band amplification was obtained only when Cas9mRNA, sgRNA, and pad5F35-sgTRAC-CAR-HDR vector plasmids were transfected simultaneously (fig. 2). The PCR amplification products were sequenced and the results demonstrated that CAR-HDR (i.e., the gene of interest CD19-CD22 CAR and two homology arms) was specifically inserted into the TRAC site in 293T cell genomic DNA.

The above results indicate that the pAd5F35-sgTRAC-CAR-HDR vector plasmid can effectively mediate site-specific site-directed gene insertion.

Example 3

Packaging adenoviruses

pAd5F35-sgTRAC-CAR-HDR and pAd5F35-TRAC-CAR-HDR vector plasmids were extracted from the bacterial suspension using the NucleoBond Xtra Midi Plus EF kit (German MN (MACHEREY-NAGEL), cat # 740422.1) according to the manufacturer's recommendations.

Mu.g of the vector plasmid was mixed with 120. mu.l of Transfection Reagent X-tremeGENE HP DNA Transfection Reagent (Roche, cat # 06366546001) in 3ml of opti-MEM (Gibco, cat # 31985070) and left at room temperature for 15min, and 293A cells (50-70% confluency) were added dropwise. After 6h, fresh DMEM complete medium was replaced. After observing the formation of plaques by placing the cell culture flask under a microscope, the culture broth was centrifuged at 500g for 10 min at 4 ℃ to remove most of the supernatant, leaving only 2 ml of supernatant to resuspend the cell pellet and storing at-80 ℃. Repeatedly freezing and thawing the virus for 3 times, infecting 293A cell again by the above method, centrifuging at 4 deg.C and 500g for 10 min to obtain cell precipitation suspension, and storing at-80 deg.C. After the infection is carried out for three times in such a cycle, the virus is greatly amplified. Thereafter, the virus was purified in vitro using an adenovirus purification kit (Biomiga, cat # V1160-01) to obtain pAd5F35-sgTRAC-CAR-HDR adenovirus and pAd5F35-TRAC-CAR-HDR adenovirus.

Example 4

Site-directed gene insertion using adenovirus

1. Site-directed gene insertion in a549 cells.

A549 cells were infected with pAd5F35-sgTRAC-CAR-HDR and pAd5F35-TRAC-CAR-HDR adenovirus. After 1 day, infected A549 cells were transfected by electroporation with sgRNA (SEQ ID NO: 5) and Cas9mRNA (SEQ ID NO: 6) targeting the TRAC gene, or Cas9mRNA alone. Adenovirus-uninfected a549 cells were transfected with sgRNA and Cas9mRNA targeting the TRAC gene, or Cas9mRNA alone as controls. After 3 days of transfection, genomic DNA of A549 cells was extracted, and the gene insertion was verified by PCR amplification. The primers used for PCR amplification were: PCR-F (SEQ ID NO: 7) on genomic DNA and PCR-R (SEQ ID NO: 8) on a vector plasmid.

And (3) performing gel electrophoresis on the PCR amplification product, and displaying that: neither transfection of Cas9mRNA and sgRNA simultaneously, nor transfection of Cas9mRNA alone, the target band was not obtained with the pAd5F35-TRAC-CAR-HDR adenovirus. Only when Cas9mRNA, sgRNA and pad5F35-sgTRAC-CAR-HDR adenovirus were transfected simultaneously, two target bands of approximately 1000bp and 2000bp in length, respectively, were obtained (fig. 3). Sequencing the two target bands, and finding that the target band with the size of 1000bp is a TRAC site which is specifically and correctly inserted into the A549 cell genome DNA by CAR-HDR and is the result of HR-mediated site-specific gene insertion; and a target band with the size of 2000bp shows that the CAR-HDR fragment is integrated into the editing site of the TRAC by the cell in a mode independent of homologous recombination, and because different indels appear in CRISPR editing, Sanger sequencing shows that a set peak appears at the combination position of the CAR-HDR sequence and the genome, which indicates that the target band is the result of the fixed-point gene insertion mediated by NHEJ.

The results show that the pAd5F35-sgTRAC-CAR-HDR adenovirus can effectively mediate the site-directed insertion of a target gene.

2. Site-directed gene insertion in T cells

In general, gene site-directed insertion of T cells is more difficult than other cells. Thus, we also tested the site-directed insertion in T cells using the methods of the invention.

Will be CTS with DynaBeads CD3/CD28^TM(Gibco, cat No. 40203D) activated T cells opti-MEM (Gibco, cat No. 31985070) were washed twice and then infected with pAd5F35-sgTRAC-CAR-HDR adenovirus.

After 1 day, T cells were transfected by electroporation with sgRNA targeting the TRAC gene (SEQ ID NO: 5) and Cas9mRNA (SEQ ID NO: 6), or Cas9mRNA alone. Untransfected infected T cells were used as controls. After 3 days of transfection, genomic DNA of T cells was extracted and gene insertion was verified by PCR amplification. The primers used for PCR amplification were: PCR-F (SEQ ID NO: 7) on genomic DNA and PCR-R (SEQ ID NO: 8) on a vector plasmid.

And (3) performing gel electrophoresis on the PCR amplification product, and displaying that: target band amplification was obtained only when Cas9mRNA, sgRNA, and pad5F35-sgTRAC-CAR-HDR adenovirus were transfected simultaneously (fig. 4). Sequencing the PCR amplification product, and the result proves that the CAR-HDR is specifically inserted into the TRAC site in the T cell genome DNA.

The above results indicate that pAd5F35-sgTRAC-CAR-HDR adenovirus can effectively mediate site-specific site-directed gene insertion into T cells.

Example 5

Site-directed gene insertion using adenovirus

To reduce genomic diversity due to NHEJ-mediated insertion, and to avoid the occurrence of non-HR-mediated insertion, we used an adenovirus containing only one sgRNA target sequence.

Specifically, as described in examples 1 and 3, a pAd5F35-sgTRAC-CAR-HDR1 adenovirus was prepared, which in turn comprises: the sgRNA-4 target sequence (SEQ ID NO: 9), the first homology arm (SEQ ID NO: 2), the gene of interest CD19-CD22 CAR (SEQ ID NO: 3), and the second homology arm (SEQ ID NO: 4).

Cas9mRNA (SEQ ID NO: 6) was then transfected into A549 cells by electroporation and infected with pAd5F35-sgTRAC-CAR-HDR1 adenovirus. Thereafter, A549 cells were co-transfected with TRAC-sgRNA (SEQ ID NO: 5) targeting the host cell genome and sgRNA (SEQ ID NO: 10) targeting the sgRNA-4 target sequence, or A549 cells were transfected with only TRAC-sgRNA (SEQ ID NO: 5) targeting the A549 host cell genome as a control. After 3 days of transfection, genomic DNA of A549 cells was extracted, and the gene insertion was verified by PCR amplification. The primers used for PCR amplification were: PCR-F (SEQ ID NO: 7) on genomic DNA and PCR-R (SEQ ID NO: 8) on a vector plasmid.

And (3) performing gel electrophoresis on the PCR amplification product, and displaying that: when the adenovirus genome was cleaved with a single sgRNA target sequence, a band of around 1000bp was obtained and there was essentially no band of around 2000bp (fig. 5). Sequencing the PCR amplification product, and the result proves that the CAR-HDR is specifically inserted into the TRAC site in the genome DNA of the cell.

The above results indicate that a single cut on the adenovirus genome can efficiently mediate HR while avoiding NHEJ-mediated insertion.

Example 6

Comparison of plasmid and adenovirus transfection efficiencies

To compare the transfection efficiencies of the plasmids and adenoviruses, we transfected A549 cells and 293T cells with GFP plasmid and pAd5F35-GFP adenovirus, respectively, and the expression level of GFP was examined by flow cytometry after 24 hours. The results show that the GFP expression levels (expressed as mean fluorescence intensity MFI) after infection of the cells with the pAd5F35-GFP adenovirus were significantly higher than those of the cells transfected with the GFP plasmid, both for a549 cells and for 293T cells, with significant statistical differences (fig. 6).

The results show that compared with the plasmid, the adenovirus can obviously improve the efficiency of transfecting cells.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

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ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 1920

aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 1980

atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 2040

aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 2100

gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 2160

aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 2220

caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 2280

gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2340

ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 2400

aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg 2460

ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 2520

tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac 2580

cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 2640

atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc 2700

tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg 2760

attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg 2820

gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg 2880

gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg 2940

gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3000

ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc 3060

gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 3120

gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag 3180

gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc 3240

ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag 3300

atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa 3360

gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 3420

cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac 3480

ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag 3540

ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 3600

agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa 3660

aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag 3720

agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa 3780

tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat 3840

aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag 3900

cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg 3960

tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac 4020

ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc 4080

gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 4140

atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg 4200

gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aa 4242

<210>7

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ctcagcacta aggaaaagcc tccag 25

<210>8

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cgccaacttg agaaggtcaa aattc 25

<210>9

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gcgctgcttc gcgatgtacg gg 22

<210>10

<211>103

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gggcgctgct tcgcgatgta gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt 103

Claims

1. A recombinant adenovirus, characterized in that: comprises a target expression frame, a first homologous arm positioned at the 5 'end of the target expression frame, a second homologous arm positioned at the 3' end of the target expression frame and one or two sgRNA target sequences.

2. A recombinant adenovirus according to claim 1, wherein: includes a sgRNA target sequence located 5 'of the first homology arm or 3' of the second homology arm.

3. A recombinant adenovirus according to claim 1, wherein: two sgRNA target sequences are included, located at the 5 'end of the first homology arm and the 3' end of the second homology arm, respectively.

4. A recombinant adenovirus according to claim 1, wherein: also included are promoters, Inverted Terminal Repeats (ITRs), and/or packaging signals.

5. A recombinant adenovirus according to claim 1, wherein the adenovirus is Ad5, Ad5F35, Ad35, Ad55, Ad2, Ad5F11, pAdBM5, or pADCMV 5.

6. A method of gene editing comprising the steps of: using the recombinant adenovirus of any one of claims 1-5 as a donor for gene editing of a cell genome by a CRISPR/Cas system in the presence of sgrnas targeting a target gene sequence of the cell genome and sgrnas targeting a target gene sequence of the sgrnas.

7. The method of claim 6, wherein: the first homology arm and the second homology arm are respectively complementary with sequences of 300-3000bp at two ends of a target gene sequence of a cell genome.

8. The method of claim 6, wherein: the cell is a mammalian cell, hematopoietic stem cell, T cell, B cell, dendritic cell, macrophage, natural killer cell, or monocyte.

9. The method of claim 6, wherein: the sgRNA targeting the genomic target gene sequence of the cell is provided in the form of RNA, or in the form of a polynucleotide encoding the sgRNA; the sgRNA targeting the sgRNA target sequence is provided as RNA or as a polynucleotide encoding the sgRNA.

10. The method of claim 6, wherein: the Cas enzyme in the CRISPR/Cas system is Cas9 or Cpf 1.

11. The method of claim 10, wherein: the Cas protease is provided as mRNA encoding the Cas protease, or as a polynucleotide encoding the Cas protease, or as a protein.

12. Use of a recombinant adenovirus according to any one of claims 1-5 in a system, composition or kit for gene editing.

13. Use of the method for gene editing according to any one of claims 7 to 11 for gene editing of fertilized egg of animal.

14. Use of a recombinant adenovirus according to any one of claims 1-5 in the preparation of engineered T cells and compositions thereof.

15. Use of the engineered T cell prepared according to claim 14 and compositions thereof in the preparation of a medicament for the treatment of cancer, infectious disease or autoimmune disease.