CN120866305A

CN120866305A - Method for editing TRAC gene locus

Info

Publication number: CN120866305A
Application number: CN202410493983.0A
Authority: CN
Inventors: 请求不公布姓名; 黄庭晖; 张照; 陈正亮
Original assignee: Nanjing Miracle Biotechnology Co ltd
Current assignee: Nanjing Miracle Biotechnology Co ltd
Priority date: 2024-04-23
Filing date: 2024-04-23
Publication date: 2025-10-31
Also published as: WO2025223099A1

Abstract

This invention relates to a method for gene editing of the TRAC gene locus. Specifically, this invention provides a method for targeting and guiding a nuclease to efficiently cleave the sgRNA of the TRAC gene, modifying CAR-T cells using the sgRNA, obtaining CAR-T cells through this method, and related gene editing systems, reagents, and kits. The CAR-T cells of this invention have reduced immunogenicity, effectively reducing the risk of graft-versus-host disease and immune rejection, while not affecting their tumor cell killing ability.

Description

Method for editing TRAC gene locus

Technical Field

The present invention relates to the fields of genetic engineering and cell biology, in particular to improved CAR-T cell therapies, gene editing methods and related applications. In particular, the invention relates to sgrnas that have high knockdown efficiency on T cell receptor genes and are not prone to off-target, thereby reducing graft versus host disease and immune rejection risk for CAR-T cells.

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T cell therapy (CHIMERIC ANTIGEN receptor T-cell immunotherapy, CAR-T) has become the most popular and research-valued therapeutic approach in current tumor immunotherapy. However, autologous CAR-T is only suitable for patients who can extract a sufficient number of T cells and have good quality, since it requires the use of T cells of the patient themselves. Moreover, for some advanced cases or patients with defective T cell function, the number and quality of T cells may not meet the therapeutic requirements. Meanwhile, since autologous CAR-T has a long treatment period and a high price, the scope of application is greatly limited, and thus new methods are needed to be explored to overcome the defects, so that the CAR-T cell therapy is widely applied, and therefore, the general CAR-T has been generated.

Universal CAR-T is a CAR-T cell engineered from T cells of healthy donors, but may cause graft-versus-host disease (GVHD) due to differences in T cells between healthy donors and patients, i.e., allogeneic CAR-T cells may attack normal tissues of patients.

T Cell Receptors (TCRs) are complex proteins that exist on the surface of T cells, and TCRs consist of two chains, called the α and β chains, each with a variable (V) region and a constant (C) region. The variable region is responsible for specific recognition of the antigenic peptide, while the constant region is linked to the cell membrane and transmits a signal. Due to the random recombination mechanism, the variable region can generate extremely high diversity, thereby enabling T cells to recognize a large number of different antigenic peptides. The TCR associates non-covalently with CD3 to form a TCR-CD3 complex that is involved in antigen recognition by T cells. TCR recognizes antigenic peptides presented by major histocompatibility complex (Major Histocompatibility Complex, MHC) molecules on antigen presenting cells (e.g., dendritic cells). When the TCR binds to the antigen peptide-MHC complex, a series of signal transduction events will be triggered, eventually activating T cells that begin an immune response to the antigen.

Human leukocyte antigens (Human Leukocyte Antigen, HLA) are the designation of the major histocompatibility complex MHC in humans. HLA molecules, which can present antigenic peptides inside and outside the body to immune cells, such as T cells, thereby triggering an immune response. This presentation process, which allows the immune system to distinguish self from non-self antigens, is a key mechanism for maintaining the immune balance of the body, and therefore organisms often face the risks of immune rejection and graft versus host disease after receiving xenografts, involves transplanting CAR-T cells of healthy individual origin into cancer patients, exerting an anti-tumor effect, and belongs to the category of allografts.

And (3) carrying out in-vitro amplification culture on T cells from healthy individuals, carrying out slow virus transfection to obtain CAR-T cells, knocking out TCR molecules in the CAR-T cells by adopting a CRISPR/Cas9 gene editing technology, effectively reducing graft versus host disease and immune rejection risks, namely preparing the universal CAR-T, and finally, reinjecting the modified CAR-T cells into a cancer patient to play an anticancer role. Thus, there is a need to develop allogeneic CAR T cells that lack endogenous T cell receptors to prevent the occurrence of GVHD.

However, the existing sgRNA targeting the T cell receptor has the problems of high risk of off-target, low knocking-down efficiency and the like. Therefore, the invention aims to provide the sgRNA which has high gene knockout efficiency and is not easy to miss.

Disclosure of Invention

The TCR is a determinant of T cell alloreaction, and an object of the present invention is to provide a stable sgRNA which has high gene cleavage efficiency, no mutual interference, and no easy off-target, so as to inhibit the expression of TCR in T cells. It is another object of the present invention to provide an agent for preparing universal CAR-T cells that is capable of knocking out TCRs efficiently, specifically, stably, without off-target and interfering with each other.

In one aspect, the invention provides an sgRNA comprising a recognition sequence for a targeting site in the TRAC gene, said sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27. Preferably, the recognition sequence of the sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs 1 to 27. More preferably, the recognition sequence of the sgRNA is the nucleotide sequence of any one of SEQ ID NOs 1 to 27.

In one aspect, the invention provides a system or agent for modifying a T cell receptor alpha constant region (TRAC) gene in a cell, comprising a sgRNA or an expression vector for expressing the sgRNA, wherein the sgRNA comprises a recognition sequence for a targeting site in the TRAC gene. Optionally, the system further comprises a nuclease or a nucleic acid encoding the nuclease. In particular, the modification is the knockout or inactivation of the TRAC gene.

In some embodiments, the system comprises a nuclease. In some embodiments, the nuclease is caused to form a complex with the sgRNA upon application of the system.

In some other embodiments, the system comprises a vector encoding the nuclease, optionally the vector encoding the nuclease is a separate vector or the same vector as the vector for expressing the sgRNA.

In some embodiments, the targeting site is located within the first, second, third, or fourth coding exons of the TRAC gene.

In some embodiments, the recognition sequence comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27.

In some embodiments, the sgrnas further comprise crRNA and tracrRNA sequences.

In some embodiments, the nuclease is capable of cleaving and inactivating the TRAC gene under the guidance of the sgRNA, and the nuclease can be various Cas nucleases commonly used in the art, e.g., cas9, cas12 nucleases. In particular, the nuclease may be a streptococcus pyogenes Cas9 (SpCas 9) nuclease.

In some embodiments, the nuclease forms a complex with the sgRNA.

In some embodiments, the cell is a CAR-T cell, which can be obtained by engineering a T cell isolated from a subject with a CAR construct.

In one aspect, the invention provides a method of making a TRAC gene-inactivated CAR-T cell comprising contacting the CAR-T cell with any of the systems or kits disclosed herein.

In one aspect, the invention provides CAR-T cells prepared by the method. Preferably, the TRAC gene in the CAR-T cells is knocked out to have reduced immunogenicity. In some embodiments, the CAR-T cell is a universal CAR-T cell.

In one aspect, the invention provides a vector comprising a nucleotide sequence encoding the sgRNA.

In one aspect, the invention provides a kit comprising:

a first container comprising a sgRNA or an expression vector for expressing the sgRNA, wherein the sgRNA comprises a recognition sequence for a targeting site in a TRAC gene, and

Optionally, a second container comprising a nuclease or a nucleic acid encoding the nuclease.

In some embodiments, the sgRNA comprised in the kit comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27.

In one aspect, the invention provides the use of an sgRNA or system as disclosed herein in the preparation of a preparation of CAR-T cells for producing inactivation of an endogenous T cell receptor alpha constant region gene (TRAC).

Brief Description of Drawings

FIG. 1 shows how the expression of the TCR/CD3 complex of T cells was examined using flow cytometry after knockdown of the TRAC gene in several sgRNA(TRAC-sgRNA-KO1、TRAC-sgRNA-KO2、TRAC-sgRNA-KO3、TRAC-sgRNA-KO4、TRAC-sgRNA-KO9、TRAC-sgRNA-KO13、TRAC-sgRNA-KO14 and TRAC-sgRNA-KO22 (FIG. 1A), together with knockdown efficiency data for all sgRNAs to which the present invention relates (FIG. 1B).

Figure 2 shows that in vitro simulated GVHD experiments, tracko CAR-T cells (shown as ko-TRAC1, ko-TRAC13 for example) were prepared with lower reactivity to allogeneic T cells than mock CAR-T by flow through analysis.

FIG. 3 shows the killing ability of several cases of TRAC ^KO CAR-T cells in vitro against U251-luc, huh7-luc and 7860-luc cells.

FIG. 4 shows that high TRAC knockout efficiency can be obtained by gel imaging analysis of the two sgRNAs (TRAC-sgRNA-KO 1 and TRAC-sgRNA-KO 13).

FIG. 5 shows the targeting and off-target site statistics for two sgRNAs (TRAC-sgRNA-KO 13 and TRAC-sgRNA-KO 8).

Detailed Description

The materials, methods, and examples described herein are illustrative only and not intended to be limiting, as methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims.

Definition of the definition

As used herein, the "TRAC gene" or "T cell receptor alpha constant region gene" in humans refers to the coding sequence of the T cell receptor alpha gene. The TCRα constant region includes, for example, the wild type sequence identified by NCBI GenID No.28755 and functional variants thereof.

As used herein, the terms "CRISPR/Cas edit", "CRISPR/Cas gene edit", "CRISPR/Cas genome edit" or similar terms refer to techniques for modifying a DNA sequence of interest using a CRISPR/Cas system. CRISPR/Cas techniques may include methods of gene expression regulation using similar principles, such as CRISPR/Cas 9-based gene expression regulation techniques.

As used herein, the term "Cas9 nuclease," "Cas9 protein," or "Cas9" is an RNA-guided nuclease belonging to the CRISPR/Cas9 gene editing system, including Cas9 proteins or variants or fragments thereof, e.g., proteins comprising an active DNA cleavage domain of Cas9 and/or a gRNA binding domain of Cas 9. As known in the art, cas9 is a component of a CRISPR/Cas gene editing system that targets and cleaves DNA target sequences under the guidance of grnas to form DNA Double Strand Breaks (DSBs). The DNA cleavage activity of Cas9 depends on two domains, ruvC and HNH, which are responsible for cleaving two strands of DNA, respectively, with the complementary strand of the guide RNA being cleaved by RuvC domain activity and the non-complementary strand being cleaved by HNH domain activity. The two domains may be individually subjected to artificial mutation inactivation as needed to achieve single-or double-stranded cleavage.

As used herein, the term "guide RNA" or "gRNA" refers to an RNA sequence comprising a guide sequence, and optionally a tracrRNA. Common guide RNAs consist of crRNA (CRISPR RNA) and tracrRNA (trans-ACTIVATING CRRNA) sequences that form a complex by partial complementarity, wherein the crRNA contains sequences that are sufficiently complementary to the target sequence to hybridize and target the CRISPR complex to a specifically bound target sequence. The term also includes single guide RNAs (sgrnas), which contain features of both crrnas and tracrrnas. Typically, the guide sequence of the sgRNA is complementary to the target nucleic acid sequence, responsible for the initial guide RNA/target base pairing. Preferably, the guide sequence of the sgRNA is intolerant of mismatches.

As used herein, the terms "guide sequence", "recognition sequence" or "spacer sequence" are used interchangeably, which is complementary to a target site in a gene of interest. The length of the recognition sequence is typically 15 to 25 nucleotides.

As used herein, the term "CAR-T cell" refers to a T cell that expresses any one of the CAR constructs, or has introduced a nucleic acid or vector encoding the CAR construct. The polynucleotide encoding the CAR construct polypeptide can be introduced into the cell in a variety of ways, or the CAR construct polypeptide can be synthesized in situ in the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, the polynucleotide construct may be integrated into the genome of the cell using stable transformation methods. In other embodiments, transient transformation methods may be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, virus-mediated methods may be used. The polynucleotide may be introduced into the cell by any suitable method, such as recombinant viral vectors (e.g., retrovirus, adenovirus), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microprojectile bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.

The term "percent (%) identity" as used herein is defined as the percentage of nucleotides that are identical between a candidate polynucleotide sequence and a polynucleotide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Sequence alignment may be performed using various methods known in the art to determine percent identity between two polynucleotide sequences, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software.

The term "vector" as used herein refers to a recombinant nucleic acid, in particular a recombinant DNA, for expressing one or more specific nucleotide sequences, or for constructing other recombinant nucleotide sequences.

Description of the embodiments

The T cell membrane surface expresses T cell receptors responsible for recognition of antigens presented by the major histocompatibility complex (MHC, in humans called leukocyte antigen, i.e. HLA molecule). Specific binding of T cell receptors to polypeptides presented by MHC triggers a series of biochemical reactions and activates T cells via numerous accessory receptors, enzymes and transcription factors, promoting their division and differentiation, and thus are critical for the cellular immune function of the immune system.

Adoptive immunotherapy of T cells (CAR T cells) genetically modified to express chimeric antigen receptors has been used as a clinical therapy for many cancers, including B-cell malignancies (e.g., acute lymphoblastic leukemia, B-cell non-hodgkin lymphoma, acute myelogenous leukemia and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, breast cancer, gastric cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and the like.

In a method employing autologous CAR T cells, the T cells of the patient are first isolated, genetically modified to express the chimeric antigen receptor, and then reinfused to the same patient. Such autologous CAR T cells are immune tolerant, but this approach is limited by the number of cells, time, expense, etc., required to produce specific CAR T cells.

Thus, it would be advantageous to use "universal" CAR T cells prepared from T cells from third party, healthy donors. However, this CAR T cell immunotherapy is limited in part by the expression of endogenous T cell receptors on the cell surface. During allograft, lymphocytes in the graft recognize the antigen of the recipient cells, develop an immune response, attack the recipient cells, and produce Graft Versus Host Disease (GVHD). Knocking out the TRAC gene means that the T Cell Receptor (TCR) on the surface of the T cell is cleared, thereby avoiding the occurrence of GVHD.

Construction of CAR-T cells

The basic principle of the chimeric antigen receptor T cell technology (CAR-T) is that T cells are extracted from a patient body and cultured in vitro, in the culture process, the T cells of the patient body express a specific tumor antigen receptor by utilizing a genetic engineering method, and after recognizing tumor-related antigens or tumor-specific antigens, the T cells can be efficiently activated and proliferated in a large amount to release antitumor active molecules, thereby exerting a strong tumor killing effect. After the modified T cells proliferate in vitro in a large amount, the CAR-T cells are injected back into the patient, and then the cancer cells expressing the specific antigen are attacked.

The key to CAR-T therapy is engineering T cells with chimeric antigen receptors, i.e., a construct of a CAR, which generally comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen binding domain is often derived from an antigen binding fragment capable of recognizing and binding to a specific antigen, e.g. in the form of a single chain antibody variable region scFv, VHH or Fab, by selecting an appropriate antigen binding domain to recognize a cell surface marker of a target cell associated with a particular disease state, e.g. a tumor. Intracellular signaling domains are used to signal effector signal functions and direct cells to perform specialized functions (e.g., cytolytic activity or helper activity, including secretion of cytokines), and generally comprise a primary signaling domain and a costimulatory signaling domain. Primary signaling domain refers to the portion of the protein that is capable of modulating primary activation of the TCR complex, either in a stimulatory manner or in an inhibitory manner, the primary signaling domain acting in a stimulatory manner typically containing a signaling motif known as an immune receptor tyrosine-based activation motif (ITAM). The co-stimulatory signaling domain refers to the intracellular signaling domain of the co-stimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen.

The invention is not particularly limited to the antigen to which the CAR construct used to engineer T cells and its design bind. The antigen of interest to which the CAR construct binds may be selected from a variety of tumor-associated antigens or tumor-specific antigens or antigens associated with other immune disorders. By way of example and not limitation, the CAR construct may be designed to recognize any antigen of interest selected from the group consisting of: CD3, CD19, CD20, 4.1BB (CD 137), OX40 (CD 134), CD16, CD47, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, epCAM, epidermal Growth Factor Receptor (EGFR), EGFRvIII, HER2, HER3, dLL3, BCMA, sialyl-Lea, 5T4, ROR1, mesothelin, folate receptor 1, VEGF receptor, epCAM, HER2/neu, HER3/neu, G250, CEA, MAGE, VEGF, FGFR, alphaVbeta-integrin 、HLA、HLA-DR、ASC、CD1、CD2、CD4、CD5、CD6、CD7、CD8、CD11、CD13、CD14、CD21、CD23、CD24、CD28、CD30、CD37、CD40、CD41、CD44、CD52、CD64、c-erb-2、CALLA、MHCII、CD44v3、CD44v6、p97、 ganglioside GM1 GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX4Trp2, gp100, tyrosinase, muc-1, telomerase, survivin, G250, p53, CA125 MUC, lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, pgp, MCSP, epHA2, GC182, GT468 or GT512, IL-17, IL-20, IL-13, and IL-4.

In some embodiments, the CAR construct comprises an antigen binding domain in the form of an scFv. In some other embodiments, the CAR construct comprises an antigen binding domain in the form of a VHH.

In some embodiments, the primary signaling domain of the CAR construct comprises an ITAM derived from a member selected from the group consisting of tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66 d.

In some embodiments, the co-stimulatory signaling domain of the CAR construct is derived from a co-stimulatory molecule selected from CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)、DAP10.

In some embodiments, the CAR construct further comprises a linking domain or linker sequence between the antigen binding domain and the transmembrane domain and/or between the transmembrane domain and the intracellular signaling domain.

In some embodiments, the engineered CAR-T cells are obtained by transfecting immune cells with a virus comprising a CAR construct. In some embodiments, the virions for transfection are produced by transfecting cells with a plasmid encoding the CAR construct and a viral packaging plasmid. In some other embodiments, the engineered CAR-T cells are obtained by transfecting immune cells with an expression vector for the CAR construct. The CAR-T cells that can be used for engineering the CAR construct are T lymphocytes, including thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells may be T helper (Th) cells, such as T helper 1 (Th 1) or T helper 2 (Th 2) cells. The T cells may be helper T cells (HTL; CD4T cells), cytotoxic T cells (CTL; CD8T cells) or any other subset of T cells. In some embodiments, T cells may include naive T cells and memory T cells.

In some embodiments, the T lymphocytes used for engineering are isolated from Peripheral Blood Mononuclear Cells (PBMCs). Methods for separating various cell fractions from PBMCs are well known to those skilled in the art. In some embodiments, the peripheral blood mononuclear cells are isolated from a subject in need of administration of a CAR-T cell therapy.

The sgRNA mediated CRISPR/Cas system of the invention

As used herein, "sgRNA", "guide RNA" targets the TRAC gene. In some embodiments, the sgrnas of the invention comprise recognition sequences that are nucleotide sequences that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27.

The sgrnas can be used to knock down the TRAC gene and/or to prepare universal CAR-T cells. Based on the teachings of the present invention, one skilled in the art will appreciate that the sgrnas of the present invention can be used in conjunction with a variety of Cas proteins. On the basis of the sgrnas of the invention, the invention provides a gene editing system comprising said sgrnas, preferably a CRISPR/Cas system, more preferably a CRISPR/Cas9 system. At the heart of the CRISPR/Cas system are the sgrnas and Cas proteins, based on the teachings of the present invention, one skilled in the art will appreciate that the sgrnas of the present invention or expression vectors expressing the sgrnas of the present invention can be used in conjunction with various Cas proteins for use in various CRISPR/Cas systems. In some embodiments, the sgrnas of the invention or expression vectors expressing the sgrnas of the invention may be used in combination with mRNA encoding a Cas protein. In some embodiments, the CRISPR/Cas system of the present invention is used to reduce or knock out TRAC gene expression.

The sgrnas of the invention comprise recognition sequences that target the TRAC gene. The recognition sequence is typically designed to be about 20nt. In addition to the recognition sequences, the sgrnas comprise crRNA and tracrRNA sequences as frameworks, preferably the tracrRNA has a neck ring structure inside. The sgRNA may comprise a 15-25 nucleotide recognition sequence at the 5' end of the sgRNA sequence. In one embodiment, the sgRNA comprises a sgRNA recognition sequence or is a full length sgRNA sequence. The full length sgrnas comprise or consist of a sgRNA recognition sequence and a corresponding framework sequence, which may comprise a portion of a crRNA and a tracrRNA.

In a preferred embodiment, the sgRNA recognition sequences are shown in SEQ ID NOS.1-27. In a more preferred embodiment, the sgRNA of the invention comprises or is a sgRNA recognition sequence and a corresponding framework sequence (e.g.SEQ ID NO:1+SEQ ID NO: 28). In a preferred embodiment, the full-length sgRNA of the invention comprises the sequence shown in any one of SEQ ID NOS: 29-55 or is the sequence shown in any one of SEQ ID NOS: 29-55.

In addition, the sgrnas of the present invention may be modified, e.g., thio-and/or methoxy-modified, to increase the stability of the sgrnas. The sgrnas of the invention may be synthesized by in vitro transcription or by chemical methods.

Methods of making modified CAR-T cells

The TRAC molecules in the CAR T cells are knocked out through a gene editing technology, so that the CAR T cells cannot express normal TCR molecules, and the graft rejection effect of the CAR T cells is reduced. In one aspect, the invention provides a method of making a modified CAR-T cell, wherein an endogenous TRAC gene in the CAR-T cell is knocked out or inactivated. Modified CAR-T cells, which have reduced cellular immunogenicity due to engineering TCR molecules, belong to the general purpose CAR-T cells. The modified CAR-T cell is also referred to herein as a TRAC ^KO CAR-T cell because it has an inactivated, knocked out, or very low expressed TRAC gene.

In the present application, after cleavage of a target locus in TRAC by Cas9 under the guidance of sgRNA, DNA sequence insertion or base deletion or the like is introduced so that TRAC gene loses biological function. Based on the disclosure of the present application and technical knowledge in the art, a person skilled in the art knows various technical points about the selection and preparation of Cas9 proteins and transfection methods.

In some embodiments, the endogenous TRAC gene is inactivated by deletion of a nucleotide. Preferably, each allele of TRAC in the genome (e.g., diploid genome) is inactivated.

In some embodiments, the inactivating the endogenous TRAC gene comprises introducing a complex with a Cas nuclease and an sgRNA into the CAR-T cell. In some embodiments, the inactivating the endogenous TRAC gene comprises introducing the sgRNA and Cas nuclease encoding plasmid separately into the CAR-T cell. The Cas nuclease includes a Cas9 nuclease. Those skilled in the art will appreciate that the sgrnas of the present invention can be used in conjunction with a variety of Cas proteins for use in a variety of CRISPR/Cas systems, such as CRISPR/Cas9 systems, CRISPR/nCas systems, CRISPR/dCas9 systems. The Cas9 protein is a multifunctional protein, and its protein structure includes a recognition Region (REC) composed of an α -helix, a nuclease region composed of HNH domain and RuvC domain, and a PAM binding region located at the C-terminus. These two important nuclease domains RuvC and HNH can cleave the DNA complementary and non-complementary strands, respectively, of gRNA, resulting in blunt-ended DNA double strand breaks. The Cas9 protein may be mutated as desired such that single-stranded DNA breaks are formed. In some embodiments, the Cas9 protein is a wild-type Cas9. In some embodiments, the Cas9 protein is derived from a streptococcus pyogenes Cas9 protein or a staphylococcus aureus Cas9 protein. In some embodiments, the Cas9 protein induces a double strand break at the target locus of the TRAC gene.

The sgRNA has the function of accurately identifying the target gene sequence in a CRISPR/Cas9 gene editing system, and the effect can influence the editing efficiency, whether off-target occurs or not, and the like, and has a decisive effect on the effect of final gene editing. Therefore, reasonably efficient design of sgrnas is an important basis for achieving gene editing, and selection of appropriate recognition sequences is the core effort of sgRNA design. For designed sgrnas, analysis can be performed based on aspects of specificity score, cleavage efficiency score, potential off-target condition, off-target site information, etc., to select the best sgrnas.

Evaluation of TRAC Gene editing Effect

The invention also provides a method for evaluating TRAC gene editing effect of modified CAR-T cells. In some embodiments, the method is performed by contacting the modified CAR-T cell with a heterologous immune cell, thereby assessing the resistance of the modified CAR-T cell to the heterologous immune cell (e.g., the viability of the CAR-T cell after contact). In some embodiments, the immune cell is allogeneic to the CAR-T cell, e.g., the immune cell comprises MHC-I that is different from MHC-I of the CAR-T cell. In some embodiments, the immune cells are isolated from PBMCs of a subject different from the subject from which the CAR-T is obtained. In some embodiments, the immune cells are selected from T cells, such as cytotoxic cd8+ T cells or natural killer cells.

In some embodiments, the methods identify TRAC gene efficiency by combining a PCR reaction with T7E1 cleavage. The method comprises the steps of extracting genome DNA of modified CAR-T cells, designing primers for PCR amplification to obtain PCR products with knockout sites, adding T7E1 enzyme for enzyme digestion, and then carrying out agarose gel electrophoresis on the reaction products, and determining the editing effect through the existence of bands.

The alpha constant region encoded by the T cell receptor alpha constant region gene is necessary for the assembly of the endogenous TCR complex on the cell surface. Thus, use of sgrnas described herein that target T cell receptor alpha constant region genes results in reduced cell surface T cell receptor expression and/or knockout. In one aspect, an sgRNA targeting a T cell receptor alpha constant region gene described herein increases the efficiency of modification of a human TCR alpha constant region gene, e.g., reduced 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up to 100% expression of an endogenous T cell receptor (e.g., an alpha/beta T cell receptor) on the cell surface of a genetically modified CAR T cell as compared to a control cell.

The invention also provides methods of assessing tumor killing effect of modified CAR-T cells. The method can assess the killing of a tumor cell by the modified CAR-T cell by incubating the modified CAR-T cell with the tumor cell at a ratio. In some embodiments, the tumor cell is a liver cancer cell, a breast cancer cell, a kidney cancer cell, a lung cancer cell, or the like.

The invention also provides for assessing off-target probability of a designed sgRNA by the Guide seq method. The Guide-seq method is a commonly used extracellular detection method for assessing off-target conditions, which uses the DNA repair mechanism of NHEJ to join a double-stranded short nucleotide sequence (dsODN) to a DNA double-strand break caused by CRISPR, equivalent to joining a first round of linker, then breaking the genome normally, and joining a second round of linker on the other side. By constructing the library in this way, sequences on one side of the off-target site can be obtained, the easier it is to ligate the sites of the dsODN, the higher the probability of off-target cleavage.

Systems, reagents and kits for gene editing

In one aspect, provided herein are modified CAR-T cells prepared by any one of the methods provided herein. In particular, an improved universal CAR-T cell is provided in which the TRAC gene is not expressed or is underexpressed. The universal CAR-T cell provided by the invention can effectively reduce graft versus host disease and immune rejection risks, thereby improving the treatment effect of the CAR-T cell.

In one aspect, provided herein are pharmaceutical compositions comprising any one of the modified CAR-T cells provided herein and a pharmaceutically acceptable carrier.

In one aspect, provided herein are compositions comprising at least one sgRNA and a nuclease or an mRNA encoding a nuclease.

In one aspect, provided herein is a gene editing system for inactivating an endogenous TRAC gene in a cell. In some embodiments, the system comprises a nuclease capable of cleaving a targeting site in an endogenous TRAC gene in the genome of a cell or a nucleic acid encoding the nuclease. In some embodiments, the system comprises an sgRNA having a recognition sequence complementary to a sequence of interest in a TRAC gene, said sgRNA being adapted to inactivate an endogenous TRAC gene. In some embodiments, the sgRNA comprises a targeting sequence of a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27.

In some embodiments, the nuclease comprises a Cas nuclease. In some embodiments, the Cas nuclease comprises a Cas9 nuclease.

The gene editing system of the invention is particularly suitable for gene editing in CAR-T cells. In some embodiments, the gene editing system of the invention is used to knock out the TRAC gene in CAR-T cells.

Exemplary embodiments:

1. One or more sgrnas comprising a recognition sequence for a targeting site in a TRAC gene, said recognition sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs 1-27.

2. The sgRNA of embodiment 1, wherein the recognition sequence has or consists of the nucleotide sequence of any one of SEQ ID NOs 1 to 27.

3. The sgRNA of embodiment 1, wherein the sgRNA further comprises a framework sequence, preferably the sequence shown as SEQ ID NO. 28.

4. The sgRNA of any of embodiments 1 to 3, wherein the sgRNA comprises or consists of the sequence set forth in any of SEQ ID NOs 29 to 55.

5. A system for modifying expression of an endogenous T cell receptor alpha constant region (TRAC) gene in a cell, comprising:

the sgRNA or vector for expressing the sgRNA of any of embodiments 1 to 4, and optionally a nuclease or a nucleic acid encoding the nuclease.

6. The system of embodiment 5, wherein the nuclease is capable of inactivating the TRAC gene under the guidance of the sgRNA, optionally wherein the nuclease is a Cas nuclease, preferably a Cas9 nuclease or a Cas12 nuclease, more preferably a spCas9 nuclease.

7. The system of embodiment 6, wherein the nuclease forms a complex with the sgRNA.

8. The system of embodiment 5 or 6, wherein the system comprises a vector encoding the nuclease, optionally the vector encoding the nuclease is a separate vector or the same vector as the vector for expressing the sgRNA.

9. The system of any of embodiments 5-8, wherein the cell is a cell that can be used for allogeneic cell therapy, optionally the cell is a T cell, e.g., a CAR-T cell.

10. A method of preparing a TRAC gene inactivated cell, the method comprising contacting the cell with the sgRNA of any one of embodiments 1 to 4 or the system of any one of embodiments 5 to 9, and introducing the sgRNA or the system into the cell, optionally, inactivating an endogenous TRAC gene in the cell.

11. The method of embodiment 10, further comprising the steps of:

1) obtaining a cell, 2) modifying the cell to encode a chimeric antigen receptor for an antigen of interest, and 3) editing a TRAC gene using the sgRNA of any one of embodiments 1-4 or the system of any one of embodiments 5-9, thereby preparing a TRAC gene-inactivated cell,

Wherein the order of step 2) and step 3) may be interchanged or step 2) and step 3) may be performed simultaneously.

12. The method of embodiment 10, wherein the nuclease in the system forms a complex with the sgRNA prior to contacting.

13. The method of embodiment 10 or 11, wherein the method further comprises introducing the sgRNA or system into the cell by electrotransfection.

14. The method of any one of embodiments 10 to 13, wherein the cell is a cell that can be used for allogeneic cell therapy, e.g., a T cell, preferably a CAR-T cell.

15. A cell prepared by the method of any one of embodiments 10 to 14.

16. A nucleic acid molecule encoding the sgRNA of any one of embodiments 1-4.

17. A vector comprising a nucleotide sequence encoding the sgRNA of any of embodiments 1-4, optionally the vector is a DNA vector such as a plasmid or a viral vector such as a retrovirus, adeno-associated virus, and lentiviral vector.

18. A kit, comprising:

a first container comprising the sgRNA of any one of embodiments 1-4 or an expression vector for expressing the sgRNA.

19. The kit of embodiment 18, further comprising a second container comprising a nuclease or a nucleic acid encoding the nuclease.

20. The kit of embodiment 18, wherein the vector for expressing sgrnas further comprises a nucleic acid sequence encoding a nuclease.

21. Use of the cell of embodiment 15 in the manufacture of a medicament for treating cancer, an autoimmune disease, or an inflammatory disease in an allogeneic subject.

22. Use of the sgRNA of any of embodiments 1-4 or the system of any of embodiments 5-9 in the preparation of T cells, preferably CAR-T cells, inactivated by an endogenous T cell receptor alpha constant region (TRAC) gene.

The beneficial effects of the invention are that

1) The sgRNA for preparing the universal CAR-T cells provided by the invention has the advantages of high knocking-down efficiency, good specificity, difficult off-target, stable targeting of TRAC, high cutting efficiency and great improvement of gene knocking-out efficiency.

2) The universal CAR-T cell provided by the invention can effectively reduce graft versus host disease and immune rejection risks, thereby improving the treatment effect of the CAR-T cell.

Examples

The following examples are provided for a better understanding of the present invention and are not intended to be limiting.

Instrument and reagent

	Merchants or sources
		X-VIVO culture medium	Lonza
DMEM medium	Gibco
		Electrical switching instrument	Lonza
PEI 40K transfection reagent	Polysciences
		PBMC	Heyou Sheng
Cas9 proteins	Kai cuo organisms
		sgRNA	Universal living things
Isolation of antibodies	Stemcell
		Separating magnetic beads	Stemcell
Activating magnetic beads	Gibco
		CD3 positive magnetic bead	Meitian and gentle
APC Anti-CD3 antibodies	Biolegend
		pMDLg/pRRE	Addgene,12251
pRSV-Rev	Addgene,12253
		pMD2.G	Addgene,12259

Example 1:T cell sorting experiments

A freezing tube containing 5 x 10 ⁷ PBMC cells was removed from the liquid nitrogen tank and thawed in a37 ℃ water bath. A new sterile cryopreservation tube was placed in advance in a magnetic rack and 1ml of X-VIVO medium was added. Then, a 15ml centrifuge tube is taken, 4ml of X-VIVO culture medium is added in advance, the thawed PBMC cells are transferred into the 15ml centrifuge tube, the centrifugation is carried out (500 Xg, 5 min), 50 mu l of STEMCELL EASYSEP separation antibody and 800 mu l of X-VIVO culture medium are added after the supernatant is discarded, the incubation is carried out for 5min, 50 mu l of separation magnetic beads are added, the mixture is added into a prepared freezing tube after the suspension is blown, and the mixture is incubated for 3min.

After incubation was completed, a 15ml centrifuge tube was taken, 7ml of medium was added, cells in the frozen tube were aspirated, and 500 Xg was added to the centrifuge tube for centrifugation for 5min and counted. After completion of the counting, the mixture was centrifuged (500 Xg, 5 min), and the supernatant was discarded, and 100. Mu.l of activated magnetic beads and 50. Mu.l of medium were added to 1X 10 ⁷ cells according to the counting result. After the cells are resuspended by blowing for a plurality of times, the cells are put into an incubator for incubation for 7min, and after the incubation is finished, the cells are again blown for incubation, and the process is repeated for three times. After the incubation was completed, the cells were cultured in T25 flasks at a ratio of 2.5×10 ⁶ cells/ml medium.

T cells after overnight culture were used for lentiviral transfection the next day in time to construct CAR-T cells.

EXAMPLE 2 construction of CAR-T cells

293T cells for viral packaging were cultured in a cell incubator at 37℃with 5% CO ₂. The medium used was DMEM medium containing 10% gibco foetal calf serum. The day before formal virus packaging, the cultured 293T cells were passaged in T75 flasks at a cell number of 1X 10 ⁷. Lentiviral packaging begins when 293T cells reach 70-80% confluence and are evenly distributed in the flask.

A1.5 ml sterile centrifuge tube was prepared, 460. Mu.l of serum-free DMEM medium was added thereto, 40. Mu.l of PEI transfection reagent was added thereto, thoroughly mixed and incubated for 5min. A1.5 ml sterile centrifuge tube was prepared, 15. Mu.l of the CAR plasmid (TRANSFER PLASMID was replaced by EF1a promoter by software on the basis of pCDH-CMV (addgene: 72265) plasmid), 5. Mu. gpMDLg/pRRE plasmid, 5. Mu.g pMD2.G plasmid and 5. Mu.g pRSV-Rev plasmid were added thereto, 470. Mu.l serum-free DMEM medium was added thereto, and the mixture was thoroughly mixed. Adding the culture medium with PEI transfection reagent into the culture medium with plasmid to form a transfection system, mixing thoroughly, and incubating for 15min. The overnight cultured 293T cells were removed, the medium in the flask was discarded, 9ml of serum-free DMEM medium was carefully added, and 1ml of the transfection system was added, and the cells were returned to the incubator for further culture for 6 hours. After 6h, the flask was removed, the medium was discarded, and 15ml of serum-free DMEM medium was carefully added. After 42h, the virus solution in the flask was collected and 15ml of serum-free DMEM medium was added again. After 24h, the virus solution in the flask was collected again, 30ml of the virus solution collected twice was poured into a 50ml sterile syringe, filtered through a 0.45 μm filter membrane, and filtered into a sterile ultracentrifuge tube. In an ultracentrifuge, the tube was centrifuged at 21,000XG for 2 hours, the supernatant was discarded, and the viral pellet was resuspended in 200. Mu. l X-VIVO medium and stored overnight at 4 ℃.

1X 10 ⁶ T cells isolated the day before were removed from the incubator, added with 100. Mu.l of the virus heavy suspension and incubated in the incubator at 37℃for 24 hours. After 24 hours, all cells are sucked, and the culture is continued by changing the liquid. The cultured cells are the desired CAR-T cells.

Example 3 electric knock-out experiments

And (3) preparing an electrotransfer liquid, namely adding all the replenishing liquid into the dissolving liquid, wherein the ratio of the dissolving liquid to the replenishing liquid is 4.5:1. An appropriate amount of medium was prepared to be placed in an orifice plate and pre-warmed in an incubator. TRAC-sgRNA was dissolved as a 100 pmol/. Mu.l solution. The TRAC-sgRNA inclusion TRAC-sgRNA-KO1、TRAC-sgRNA-KO2、TRAC-sgRNA-KO3、TRAC-sgRNA-KO4、TRAC-sgRNA-KO5、TRAC-sgRNA-KO6、TRAC-sgRNA-KO7、TRAC-sgRNA-KO8、TRAC-sgRNA-KO9、TRAC-sgRNA-KO10、TRAC-sgRNA-KO11、TRAC-sgRNA-KO12、TRAC-sgRNA-KO13、TRAC-sgRNA-KO14、TRAC-sgRNA-KO15、TRAC-sgRNA-KO16、TRAC-sgRNA-KO17、TRAC-sgRNA-KO18、TRAC-sgRNA-KO19、TRAC-sgRNA-KO20、TRAC-sgRNA-KO21、TRAC-sgRNA-KO22、TRAC-sgRNA-KO23、TRAC-sgRNA-KO24、TRAC-sgRNA-KO25、TRAC-sgRNA-KO26、TRAC-sgRNA-positive control. is shown in Table 1 below.

TABLE 1 TRAC-sgRNA recognition sequences

In the present application, each full-length sgRNA sequence is equal to the recognition region sequence of the corresponding sgRNA+the framework sequence shown in SEQ ID NO. 28 (from 5 'end to 3' end), i.e., each full-length sgRNA sequence consists of the recognition region sequence of the corresponding sgRNA and the framework sequence shown in SEQ ID NO. 28 from 5 'end to 3' end.

TRAC-sgRNA was mixed with 24pmol of Cas9 protein in an amount of 72pmol each. Incubate for 10min. After centrifugation to count the cells, 1X 10 ⁶ cells were re-centrifuged and resuspended using 20. Mu.l of electrotransfer fluid.

Mu.l of resuspended cell fluid was added to each RNP. The cell fluid was transferred into the strip. The electrotransport instrument was turned on and the slat option was selected, electrotransport knockout was performed using the T cell editing program CM 119. The wells to which the cell fluid has been added are selected, and the T CELLEDITING option is selected. The start button is turned on, and after the completion of the electric transfer, the mixed solution is transferred to a medium prepared in advance and cultured in an incubator.

The knockdown cells were resuspended in 100 μl of PBS after centrifugation, 5 μl of APC Anti-CD3 antibody was added, incubated at 4℃for 30min, the supernatant was discarded after centrifugation and resuspended in 200 μl of PBS, and the up-flow cytometer was used to determine the knockdown efficiency of CD3, thereby assessing the knockdown efficiency of the T cell receptor, and as shown in FIG. 1, part of the screened sgRNAs gave CAR-T cells with high TRAC knockdown rate.

EXAMPLE 4 GVHD experiment

A freezing tube containing 5 x 10 ⁷ PBMC cells was removed from the liquid nitrogen tank and thawed in a 37 ℃ water bath. Taking a clean 15ml centrifuge tube, adding 9ml of culture medium, transferring the thawed cells into the centrifuge tube, centrifuging at 500 Xg for 5min, discarding the supernatant, adding 10ml of culture medium, and fully blowing and uniformly mixing.

Cell suspension and trypan blue 1:1 were mixed, cell concentration was measured by a cytometer, sufficient PBMC cells were aspirated, centrifuged (500×g,5 min), the supernatant discarded, the cells resuspended in 25ml medium, and transferred to a T75 cell culture flask. PBMC cells are irradiated with 20gy units by using ELEKTA INFINITY linear accelerator to enable the PBMC cells to be in a death or pseudodeath state, 1X 10 ⁶ PBMC subjected to radiation are taken as target cells, then 1X 10 ⁶ CAR-T cells are taken as effector cells to be incubated with the PBMC, and the depletion and activation of the CAR-T cells are detected in an up-flow manner after 12 hours.

As shown in fig. 2, the prepared tracko CAR-T cells (shown as ko-TRAC1, ko-TRAC13 for example) have lower reactivity to allogeneic T cells than mock CAR-T by flow analysis.

Example 5 TRAC knockdown CAR-T cell mediated tumor cell killing

The X-VIVO serum-free cell culture medium was placed in a 37 ℃ water bath for pre-heating in advance. 7860-luc, U251-luc and Huh7-luc cells in good preparation. Before plating and killing, transferring the culture medium in 7860-luc, U251-luc and Huh7-luc cells into a 15ml centrifuge tube, rinsing the bottom of a culture bottle with PBS, adding a proper amount of 0.25% pancreatin for digestion, suspending cells, sucking the culture supernatant of the primary cells by a pipette, adding the culture bottle for stopping digestion, blowing off the cells, transferring the cells into a 15ml centrifuge tube, and centrifuging (400 Xg, 5 min) to remove the supernatant. In a 96-well cell culture plate, 7860-luc, U251-luc and Huh7-luc cells were added at a concentration of 3.33X10- ⁵ cells/ml per well in 60. Mu.l so that 20000 cells were added per well. And placing the cell culture plate paved with target cells at 37 ℃ and incubating for 3-5 h in a 5% carbon dioxide incubator.

The CAR-T cells to be tested regulate the suspension concentration of the cells according to different positive rates and effective target ratios E:T. When the target cells were 20000 cells and the effective target ratio ET was 8:1, the amount of the medium added was 60. Mu.l, and therefore, the cells were adjusted to (160000/0.06/positive rate) cells/ml. At the same time, the 1:1, 1:2, 1:4 effective target ratios were sequentially half-diluted (150. Mu.l cell suspension+150. Mu.l X-VIVO serum-free cell culture medium with 10% FBS).

Cell culture plates with cells spread are placed at 37 ℃ and incubated for 8h in a 5% carbon dioxide incubator. The reagents in the ONE-Glo Luciferase ASSAY SYSTEM kit were removed from the-20℃refrigerator before the incubation was completed and left at room temperature until the reagents melted. According to the instruction, E606A powder was dissolved with E605A reagent, and after dissolution was completed, the mixture was packaged in EP tube and stored in a-20℃refrigerator.

And opening the multifunctional microplate reader and software, selecting a luminencemode, and performing plate reading layout. Adding 100 μl of prepared reagent into each cell, blowing, mixing, standing at room temperature in dark for 10min, and transferring 180 μl of solution in the cell culture plate into 96-well white flat bottom plate by pipetting gun to avoid air bubbles. And placing the 96-hole white flat bottom plate with an enzyme label instrument for reading data, and deriving and storing the data for calculating the cell killing rate. Cell killing = (background luminescence value-sample luminescence value)/background luminescence value 100%.

As shown in fig. 3, the knocked-out CAR-T cells have a similar killing ability to mock CAR-T cells, and cannot lose the killing ability due to knocking-out, and meanwhile, the knocked-out CAR-T cells also have no killing ability to non-target cells, so that the prepared knocked-out CAR-T cells have good specific tumor killing ability.

EXAMPLE 6T 7E1 enzyme assay

5X 10 ⁶TRAC^ko CAR-T cells were prepared, centrifuged at 250 Xg for 5min, the supernatant discarded and the cells resuspended in 200. Mu.l PBS. The genome of the cell is extracted byGenomic DNA kit. Mu.l of Proteinase K and 20. Mu.l of RNase A were added to the cells, and after brief vortexing, incubated for 2min at room temperature. 200 μl was addedAfter vortexing the genome lysis/binding buffer again, incubation was performed for 10min at 55 ℃. 200. Mu.l of absolute ethanol was added to the lysate and stirred for 5 seconds to form a homogeneous solution.

Adding the homogeneous solution toThe column was centrifuged at 10000 Xg for 1min. After centrifugation, the separation column is taken out and put into cleanIn the collection tube. To the column 500. Mu.l Wash Buffer 1 in ethanol was added and the mixture was centrifuged at 10000g for 1min. After centrifugation, the separation column is taken out and put into cleanIn the collection tube. 500 μl Wash Buffer 2 in ethanol was added to the column and centrifuged at 20000g for 3min. The column was placed in a sterile 1.5ml centrifuge tube and 100. Mu.l was added to the columnGenome elution buffer. After incubation for 1min at room temperature, 20000 was centrifuged for 1min. A new sterile 1.5ml centrifuge tube was taken, the column was placed therein, and 100. Mu.l of the solution was added to the columnGenome elution buffer. After incubation for 1min at room temperature, 20000g was centrifuged for 1.5min.

The DNA concentration is quantified by using a micro ultraviolet spectrophotometer, 200ng of DNA is extracted to carry out PCR amplification by using high-fidelity DNA polymerase after the DNA concentration is regulated, and a PCR product with a knockout site is obtained. One example of the experimental primers is shown in Table 2.

TABLE 2 TRAC amplification primer sequences

All the above operations were performed on the non-knocked-out CAR-T in the same manner, and PCR products of the non-knocked-out group were obtained. The PCR products were purified using the bi-yun DNA purification kit. Adding an equal volume of DNA purification binding solution into the PCR product, and uniformly mixing. The homogeneous solution was added to a DNA purification column and after incubation for 1min 20000 Xg was centrifuged for 5min. After centrifugation, the liquid in the collection tube was discarded. 700. Mu.l of the washing solution was added to the DNA purification column, and after incubation for 1min, 20000 Xg was centrifuged for 1min. After centrifugation, the liquid in the collection tube was discarded. A further 500. Mu.l of the washing solution was added to the DNA purification column, and the mixture was centrifuged at 20000 Xg for 1min. Further washing off impurities and discarding the liquid in the collecting pipe. The solution was centrifuged again to remove the remaining liquid and the remaining ethanol was fully evaporated. The DNA purification column was placed on a 1.5ml centrifuge tube, and 50. Mu.l of the eluent was added to the center of the column surface in the tube, so that the liquid was absorbed by the purification column, and left for 1min. After centrifugation for 1min at 20000 Xg, the resulting liquid was high purity DNA. T7E1 experiments used the Norpran T7 Endonuclease I kit. The T7E1 cleavage reaction system was prepared as shown in Table 3.

TABLE 3T 7E1 cleavage reaction System

After preparing a T7E1 enzyme digestion reaction system, carrying out annealing reaction. The annealing procedure is shown in table 4.

TABLE 4T 7E1 cleavage reaction annealing procedure

Temperature (temperature)	Time of
		95°C	5min
95~85°C	-2°C/sec
		85~25°C	-0.1°C/sec
4°C	∞

To the annealed product 1. Mu.l of T7E1 was added and incubated at 37℃for 30min. The cleavage reaction was stopped by adding 1.5. Mu.l of 0.25 MEDTA. The digested products were directly detected by 2% agarose gel electrophoresis.

As shown in fig. 4, by analysis of gel imaging, it was found that a high TRAC knockout efficiency was obtained for a portion of the screened sgrnas.

Example 7 guide-seq

Cell culture and molecular knife transfection cells were cultured and CRISPR/Cas9 gene scissors and dsODN tag were transfected into cells. dsODN tag is a DNA fragment containing a primer and a barcode sequence for labeling the DNA sequence cleaved by Cas 9. The purpose of this step is to guide the Cas9 protein within the cell to target a specific DNA sequence and label the cleaved DNA sequence.

DNA extraction genomic DNA is extracted from the cells, the purpose of this step being to obtain a DNA sequence cleaved by Cas9 and to carry out subsequent banking.

And (3) PCR amplification: PCR amplification was performed using forward ODN primers and reverse ODN primers. The dsODN tag primer is ligated to the DNA sequence cleaved by Cas9 and a sufficient number of DNA fragments are amplified for subsequent sequencing analysis.

And second generation sequencing, namely performing high-throughput sequencing on the DNA fragments amplified by PCR, and determining the targeting position of the Cas9 primer by determining the barcode sequence and the corresponding targeting sequence of each primer. After sequencing, the data were further analyzed.

Splitting samples because the GUIDE-seq library building process uses the barcode sequence, the sequencing reads of each sample need to be split according to the barcode sequence. It is necessary to separate the sequencing data of each sample for subsequent analysis.

PCR repetition is removed, in which the same DNA fragment may be amplified multiple times during PCR amplification, resulting in PCR repetition. In order to avoid such effects, it is necessary to perform deduplication processing on the sequencing data, reduce the deviation introduced by PCR amplification, and improve the accuracy and reliability of data analysis.

Alignment the re-processed sequencing data is aligned with the reference genome to determine the targeting sequence of each primer, thereby identifying the DNA sequence cleaved by Cas9 and accurately determining its targeting position.

And (3) identifying candidate sites and off-target sequences, namely identifying the candidate sites and the off-target sequences according to the comparison result. Candidate sites refer to targeting sequences that are cleaved by Cas9, and off-target sequences refer to DNA sequences that are similar to the targeting sequences but that are not cleaved by Cas 9. The purpose of this step is to assess the specificity and accuracy of the CRISPR system and to determine possible site mutations or insertions/deletions etc.

Reporting, namely sorting the identified sites according to the number of reads and annotating. And summarizing and reporting the analysis results, describing information about Cas9 targeting sites and off-target sequences.

And (3) visualizing the detected sequences at the target and off-target sites, for example, performing visual display by using IGV and other software, and obtaining visual result display so as to better understand the analysis result.

As shown in the results of FIG. 5, the designed sgRNA sequence has no mismatch rate, low off-target probability and high safety.

While the application has been shown and described with respect to exemplary embodiments thereof, it will be understood by those skilled in the art that the foregoing embodiments are not to be construed as limiting the application, and that variations, substitutions and modifications may be made without departing from the spirit, principles and scope of the application.

Claims

2. The sgRNA of claim 1, wherein the recognition sequence has or consists of the nucleotide sequence of any one of SEQ ID NOs 1 to 27.

3. The sgRNA of claim 1, wherein the sgRNA further comprises a framework sequence, preferably the sequence shown in SEQ ID No. 28.

4. The sgRNA of any one of claims 1 to 3, wherein the sgRNA comprises or consists of the sequence set forth in any one of SEQ ID NOs 29 to 55.

the sgRNA or the vector for expressing the sgRNA of any one of claims 1 to 4, and optionally a nuclease or a nucleic acid encoding the nuclease.

6. The system of claim 5, wherein the nuclease is capable of inactivating a TRAC gene under the guidance of the sgRNA, optionally wherein the nuclease is a Cas nuclease, preferably a Cas9 nuclease or a Cas12 nuclease, more preferably a spCas9 nuclease.

7. The system of claim 6, wherein the nuclease forms a complex with the sgRNA.

8. The system of claim 5 or 6, wherein the system comprises a vector encoding the nuclease, optionally the vector encoding the nuclease is a separate vector or the same vector as the vector for expressing the sgRNA.

9. The system of any one of claims 5-8, wherein the cell is a cell that can be used for allogeneic cell therapy, optionally the cell is a T cell, such as a CAR-T cell.

10. A method of preparing a TRAC gene inactivated cell, the method comprising contacting the cell with the sgRNA of any one of claims 1-4 or the system of any one of claims 5-9, and introducing the sgRNA or the system into the cell, optionally, inactivating an endogenous TRAC gene in the cell.

11. The method of claim 10, further comprising the step of:

1) obtaining a cell, 2) modifying the cell to encode a chimeric antigen receptor for an antigen of interest, and 3) editing a TRAC gene using the sgRNA of any one of claims 1-4 or the system of any one of claims 5-9, thereby preparing a TRAC gene-inactivated cell,

12. The method of claim 10, wherein nuclease in the system forms a complex with the sgRNA prior to contacting.

13. The method of claim 10 or 11, wherein the method further comprises introducing the sgRNA or system into the cell by electrotransfection.

14. The method of any one of claims 10 to 13, wherein the cell is a cell that can be used for allogeneic cell therapy, such as a T cell, preferably a CAR-T cell.

15. A cell prepared by the method of any one of claims 10 to 14.

16. A nucleic acid molecule encoding the sgRNA of any one of claims 1-4.

17. A vector comprising a nucleotide sequence encoding the sgRNA of any of claims 1-4, optionally the vector is a DNA vector such as a plasmid or a viral vector such as a retrovirus, adeno-associated virus and lentiviral vector.

18. A kit, comprising:

A first container comprising the sgRNA of any one of claims 1-4 or an expression vector for expressing the sgRNA.

19. The kit of claim 18, further comprising a second container comprising a nuclease or a nucleic acid encoding the nuclease.

20. The kit of claim 18, wherein the vector for expressing sgrnas further comprises a nucleic acid sequence encoding a nuclease.

21. Use of the cell of claim 15 in the manufacture of a medicament for treating cancer, an autoimmune disease or an inflammatory disease in an allogeneic subject.

22. Use of the sgRNA of any one of claims 1 to 4 or the system of any one of claims 5 to 9 for the preparation of T cells, preferably CAR-T cells, inactivated by endogenous T cell receptor alpha constant region (TRAC) genes.