WO2025223099A1 - Gene editing method for trac locus - Google Patents

Abstract

A gene editing method for a TRAC locus. Specifically, provided are an sgRNA targeting the TRAC gene and guiding a nuclease to efficiently cleave the TRAC gene, a method for using the sgRNA to modify a CAR-T cell, a CAR-T cell obtained by the method, and a related gene editing system, reagent and kit. The CAR-T cell reduces immunogenicity, and can effectively reduce the risk of graft-versus-host diseases and immune rejection; and the tumor cell killing capability of the CAR-T cell would not be affected.

Description

Gene editing methods for the TRAC locus Technical Field

This invention relates to the fields of genetic engineering and cell biology, particularly to improved CAR-T cell therapy, gene editing methods, and related applications. Specifically, this invention relates to sgRNAs that have high knockdown efficiency on T cell receptor genes and are less prone to off-target effects, thereby reducing the risk of graft-versus-host disease and immune rejection in CAR-T cells.

Background Technology

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become the most popular and research-worthy treatment method in tumor immunotherapy. However, autologous CAR-T requires the use of the patient's own T cells, thus it is only suitable for patients who can obtain a sufficient number of high-quality T cells. Moreover, for some advanced cases or patients with T cell dysfunction, the quantity and quality of their T cells may not meet the treatment requirements. At the same time, the long treatment cycle and high cost of autologous CAR-T have greatly limited its application. Therefore, it is necessary to explore new methods to overcome these shortcomings in order to achieve the widespread application of CAR-T cell therapy. Thus, universal CAR-T has emerged.

Universal CAR-T is created by modifying T cells from healthy donors. However, due to the differences between T cells in healthy donors and patients, it may cause graft-versus-host disease (GVHD), in which allogeneic CAR-T cells may attack the patient's normal tissues.

The T cell receptor (TCR) is a complex protein located on the surface of T cells. The TCR consists of two chains, called the α chain and the β chain, each with a variable (V) region and a constant (C) region. The variable region is responsible for specifically recognizing antigenic peptides, while the constant region connects to the cell membrane and transmits signals. Due to random recombination mechanisms, the variable region can generate extremely high diversity, enabling T cells to recognize a large number of different antigenic peptides. The TCR binds nonvalently to CD3, forming the TCR-CD3 complex, which participates in T cell antigen recognition. The TCR recognizes antigenic peptides presented by the major histocompatibility complex (MHC) molecule on antigen-presenting cells (such as dendritic cells). When the TCR binds to the antigenic peptide-MHC complex, it triggers a series of signal transduction events, ultimately activating the T cell and initiating an immune response against the antigen.

Human leukocyte antigen (HLA) is the name for the major histocompatibility complex (MHC) in the human body. HLA molecules present antigenic peptides from inside and outside the body to immune cells, such as T cells, thereby triggering an immune response. This presentation process enables the immune system to distinguish between self and non-self antigens and is a key mechanism for maintaining immune homeostasis. Therefore, organisms that receive xenografts or allogeneic grafts often face the risks of immune rejection and graft-versus-host disease. In this technology, CAR-T cells from healthy individuals are transplanted into cancer patients to exert anti-tumor effects, which falls under the category of allogeneic transplantation.

CAR-T cells are obtained by expanding and culturing T cells from healthy individuals in vitro and then transfecting them with lentiviruses. Next, CRISPR/Cas9 gene editing technology is used to knock out the TCR molecule in the CAR-T cells, effectively reducing the risk of graft-versus-host disease and immune rejection, thus creating a universal CAR-T. Finally, the modified CAR-T cells are reinfused into cancer patients to exert their anti-cancer effect. Therefore, it is necessary to develop allogeneic CAR T cells lacking endogenous T cell receptors to prevent the occurrence of GVHD.

However, existing sgRNAs targeting T cell receptors suffer from high off-target risks and low knockdown efficiency. Therefore, this invention aims to provide an sgRNA with high gene knockdown efficiency and low off-target risk.

Summary of the Invention

TCR is a determining factor in T cell alloimmunity. One object of this invention is to provide a stable sgRNA that is highly efficient in gene cleavage, does not interfere with each other, is not prone to off-target effects, and can inhibit TCR expression in T cells. Another object of this invention is to provide a reagent for preparing universal CAR-T cells that can efficiently, specifically, stably, and without off-target effects or interference with each other to knock out TCR.

In one aspect, the present invention provides an sgRNA comprising a recognition sequence targeting a site in the TRAC gene, said sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the nucleotide sequences in SEQ ID NO: 1-27. Preferably, the recognition sequence of said sgRNA comprises a nucleotide sequence in any of SEQ ID NO: 1-27. More preferably, the recognition sequence of said sgRNA is a nucleotide sequence in any of SEQ ID NO: 1-27.

In one aspect, the present invention provides a system or reagent for modifying the T cell receptor α constant region (TRAC) gene in cells, comprising: sgRNA or an expression vector for expressing said sgRNA, wherein said sgRNA includes a recognition sequence targeting a site in the TRAC gene. Optionally, the system further comprises a nuclease or a nucleic acid encoding said nuclease. Specifically, the modification is knocking out or inactivating the TRAC gene.

In some embodiments, the system comprises a nuclease. In some embodiments, when using the system, the nuclease forms a complex with the sgRNA.

In some other embodiments, the system includes a vector encoding the nuclease, optionally a separate vector from or the same vector used to express sgRNA.

In some implementations, the target site is located in the first, second, third, or fourth coding exon of the TRAC gene.

In some embodiments, the identifying sequence comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the nucleotide sequences in SEQ ID NO:1-27.

In some implementations, the sgRNA also comprises crRNA and tracrRNA sequences.

In some embodiments, the nuclease is capable of cleaving and inactivating the TRAC gene under the guidance of the sgRNA. The nuclease can be various Cas nucleases commonly used in the art, such as Cas9 and Cas12 nucleases. Specifically, the nuclease can be Streptococcus pyogenes Cas9 (SpCas9) nuclease.

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

In some implementations, the cells are CAR-T cells, which can be obtained by engineering T cells isolated from a subject using a CAR construct.

In one aspect, the present invention provides a method for preparing TRAC gene-inactivated CAR-T cells, the method comprising: contacting the CAR-T cells with any of the systems or kits disclosed herein.

In one aspect, the present invention provides CAR-T cells prepared by the method described above. Preferably, the TRAC gene in the CAR-T cells is knocked out, thereby exhibiting reduced immunogenicity. In some embodiments, the CAR-T cells are universal CAR-T cells.

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

In one aspect, the present invention provides a kit comprising:

A first container, the first container containing sgRNA or an expression vector for expressing said sgRNA, wherein said sgRNA includes a recognition sequence targeting a site in the TRAC gene; and

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

In some embodiments, the sgRNA included in the kit comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the nucleotide sequences in SEQ ID NO: 1-27.

In one aspect, the present invention provides the use of sgRNA or systems as disclosed herein in the preparation of CAR-T cells for generating endogenous T cell receptor α constant region gene (TRAC) inactivated.

Brief description of the attached diagram

Figure 1 shows the expression of the TCR/CD3 complex in CAR-T cells after knocking out the TRAC gene with several sgRNAs (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) using flow cytometry (Figure 1A), and includes knockout efficiency data for all the sgRNAs involved in this invention (Figure 1B).

Figure 2 shows that, in an in vitro simulated GVHD experiment, flow cytometry analysis revealed that the prepared TRAC ko CAR-T cells (showing ko-TRAC1 and ko-TRAC13 as examples) exhibited lower responsiveness to allogeneic T cells compared to mock CAR-T cells.

Figure 3 shows the killing ability of several TRAC ^KO CAR-T cells against U251-luc, Huh7-luc and 7860-luc cells in vitro.

Figure 4 shows that gel imaging analysis indicates that both sgRNAs (TRAC-sgRNA-KO1 and TRAC-sgRNA-KO13) can achieve high TRAC knockout efficiency.

Figure 5 shows the statistics of target sites and off-target sites for the two sgRNAs (TRAC-sgRNA-KO13 and TRAC-sgRNA-KO8).

Invention Details

The materials, methods, and embodiments described herein are illustrative only and not limiting. Similar or equivalent methods and materials may be used in the practice or testing of this disclosure. Other features and advantages of this disclosure will become apparent from the following detailed description and claims.

definition

As used herein, the “TRAC gene” or “T cell receptor α constant region gene” in humans refers to the coding sequence of the T cell receptor α gene. The TCRα constant region includes, for example, the wild-type sequence and its functional variants as identified by NCBI Gen ID NO. 28755.

As used herein, the terms “CRISPR/Cas editing,” “CRISPR/Cas gene editing,” “CRISPR/Cas genome editing,” or similar terms refer to techniques that modify target DNA sequences using the CRISPR/Cas system. CRISPR/Cas technology may include methods that utilize similar principles to regulate gene expression, such as gene expression regulation techniques based on CRISPR/Cas9.

As used herein, the terms “Cas9 nuclease,” “Cas9 protein,” or “Cas9” refer to RNA-directed nucleases belonging to the CRISPR/Cas9 gene editing system, including the Cas9 protein or variants or fragments thereof, such as proteins containing the active DNA-cutting domain and/or the gRNA-binding domain of Cas9. As is known in the art, Cas9 is a component of the CRISPR/Cas gene editing system that, guided by gRNA, targets and cleaves DNA target sequences to form DNA double-strand breaks (DSBs). The DNA-cutting activity of Cas9 depends on two domains: RuvC and HNH, which are responsible for cleaving the two strands of DNA, respectively. Activity of the RuvC domain cleaves the complementary strand of the guiding RNA, while activity of the HNH domain cleaves the non-complementary strand. These two domains can be artificially mutated and inactivated as needed to achieve single-strand or double-strand cleavage.

As used herein, the term "guide RNA" or "gRNA" refers to an RNA sequence containing 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 through partial complementarity, wherein the crRNA contains a sequence sufficiently complementary to the target sequence to hybridize and target the CRISPR complex to the specifically binding target sequence. The term also includes single guide RNA (sgRNA), which combines the characteristics of both crRNA and tracrRNA. Typically, the guide sequence of the sgRNA is complementary to the target nucleic acid sequence and is responsible for 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 and are complementary to the target site in the target gene. The recognition sequence is typically 15 to 25 nucleotides in length.

As used herein, the term "CAR-T cell" refers to a T cell that expresses any CAR construct or has been introduced with nucleic acid or a vector encoding a CAR construct. Polynucleotides encoding CAR construct peptides can be introduced into cells using various methods, or CAR construct peptides can be synthesized in situ within the cells. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the cell's genome. In other embodiments, transient transformation methods can be used for transient expression of the polynucleotide construct, and the polynucleotide construct is not integrated into the cell's genome. In other embodiments, virus-mediated methods can be used. Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides can be included in vectors, such as plasmid vectors or viral vectors.

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

As used in this article, the term "vector" refers to recombinant nucleic acids, particularly recombinant DNA, which are used to express one or more specific nucleotide sequences or to construct other recombinant nucleotide sequences.

Implementation Method

T cell membranes express T cell receptors, which are responsible for recognizing antigens presented by the major histocompatibility complex (MHC, known as leukocyte antigens or HLA molecules in humans). The specific binding of T cell receptors to peptides presented by the MHC triggers a series of biochemical reactions and activates T cells through numerous co-receptors, enzymes, and transcription factors, promoting their division and differentiation. Therefore, T cell receptors are crucial for the cellular immune function of the immune system.

Adoptive immunotherapy using genetically modified T cells expressing chimeric antigen receptors (CAR T cells) has been used in clinical treatment for many cancers, including B-cell malignancies (such as acute lymphoblastic leukemia, B-cell non-Hodgkin lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, breast cancer, gastric cancer, mesothelioma, melanoma, prostate cancer, and pancreatic cancer.

In the autologous CAR T cell method, the patient's T cells are first isolated, genetically modified to express chimeric antigen receptors, and then reinfused into the same patient. These autologous CAR T cells exhibit immune tolerance; however, this method is limited by factors such as the amount of cells required to produce specific CAR T cells, the time involved, and the cost.

Therefore, "universal" CAR T cells prepared using T cells from third-party, healthy donors would be advantageous. However, this CAR T cell immunotherapy is partly limited by the expression of endogenous T cell receptors on the cell surface. During allogeneic transplantation, lymphocytes in the graft recognize antigens on the recipient cells, triggering an immune response that attacks the recipient cells, resulting in graft-versus-host disease (GVHD). Knocking out the TRAC gene means that the T cell receptor (TCR) on the surface of T cells is cleared, thereby preventing the occurrence of GVHD.

Construction of CAR-T cells

The basic principle of chimeric antigen receptor T-cell (CAR-T) technology is to extract T cells from the patient and culture them in vitro. During the culture process, genetic engineering is used to modify the patient's own T cells to express specific tumor antigen receptors. After recognizing tumor-associated antigens or tumor-specific antigens, the T cells are efficiently activated and proliferate in large numbers, releasing anti-tumor active molecules, thereby exerting a powerful tumor-killing effect. After the modified T cells proliferate in large quantities in vitro, the CAR-T cells are injected back into the patient to attack cancer cells expressing specific antigens.

The key to CAR-T therapy is the engineering of T cells using chimeric antigen receptor (CAR) constructs. CAR constructs typically contain extracellular antigen-binding domains, transmembrane domains, and intracellular signal transduction domains. Antigen-binding domains often originate from antigen-binding fragments capable of recognizing and binding specific antigens, such as the variable region of a single-chain antibody (scFv, VHH, or Fab). By selecting appropriate antigen-binding domains, cell surface markers associated with specific disease states, such as tumors, can be identified. Intracellular signal transduction domains are used to transduce effector and functional signals and guide cells to perform specific functions (e.g., cytolytic or co-activating activities, including cytokine secretion). These domains typically contain primary signal transduction domains and co-stimulatory signal transduction domains. Primary signal transduction domains are protein portions that can regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Stimulatory primary signal transduction domains often contain signal transduction motifs known to be based on the tyrosine-based activation motif of the immune receptor (ITAM). Co-stimulatory signal transduction domains are intracellular signal transduction domains of co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or Fc receptors, that provide a second signal required for the efficient activation and function of T lymphocytes after binding to an antigen.

This invention does not impose any particular limitations on the CAR constructs used for engineering T cells or the antigens they are designed to bind to. The target antigens bound to the CAR constructs can be selected from a variety of tumor-associated antigens, tumor-specific antigens, or antigens related to other immune diseases. For example, and not limited to, the CAR construct may be designed to recognize a target antigen selected from any of the following: CD3, CD19, CD20, 4.1BB (CD137), OX40 (CD134), 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, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, C D13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NF X2, SSX2, SSX4Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125MUC, 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 scFv. In other embodiments, the CAR construct comprises an antigen-binding domain in the form of VHH.

In some implementations, the primary signal transduction domain of the CAR construct contains an ITAM derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

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

In some embodiments, the CAR construct further includes a linker domain or adapter sequence between the antigen-binding domain and the transmembrane domain and/or between the transmembrane domain and the intracellular signal transduction domain.

In some embodiments, engineered CAR-T cells are obtained by transfecting immune cells with a virus containing the CAR construct. In some embodiments, the virion used for transfection is generated by transfecting cells with a plasmid encoding the CAR construct and a viral packaging plasmid. In some other embodiments, engineered CAR-T cells are obtained by transfecting immune cells with an expression vector of the CAR construct. CAR-T cells that can be used for CAR construct modification are T lymphocytes, including thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 T cells), cytotoxic T cells (CTL; CD8 T cells), or any other T cell subset. In some embodiments, T cells may include primordial T cells and memory T cells.

In some embodiments, the engineered T lymphocytes are isolated from peripheral blood mononuclear cells (PBMCs). Methods for isolating 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 subjects requiring CAR-T cell therapy.

The sgRNA-mediated CRISPR/Cas system of the present invention

As used herein, "sgRNA" or "guide RNA" targets the TRAC gene. In some embodiments, the sgRNA of the present invention comprises a recognition sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the nucleotide sequences in SEQ ID NO: 1-27.

The sgRNA can be used to knock down the TRAC gene and/or to prepare universal CAR-T cells. Based on the teachings of this invention, those skilled in the art will understand that the sgRNA of this invention can be used in combination with various Cas proteins. Based on the sgRNA of this invention, this invention provides a gene editing system comprising the sgRNA, preferably a CRISPR/Cas system, more preferably a CRISPR/Cas9 system. The core of the CRISPR/Cas system is sgRNA and Cas protein. Based on the teachings of this invention, those skilled in the art will understand that the sgRNA of this invention or the expression vector expressing the sgRNA of this invention can be used in combination with various Cas proteins, thereby being used in various CRISPR/Cas systems. In some embodiments, the sgRNA of this invention or the expression vector expressing the sgRNA of this invention can be used in combination with mRNA encoding the Cas protein. In some embodiments, the CRISPR/Cas system of this invention is used to reduce or knock out TRAC gene expression.

The sgRNA of this invention contains a recognition sequence targeting the TRAC gene. The recognition sequence is typically designed to be about 20 nt. In addition to the recognition sequence, the sgRNA also contains crRNA and tracrRNA sequences as a framework, preferably, the tracrRNA has a neck loop structure within it. The sgRNA may contain a 15-25 nucleotide recognition sequence at the 5' end of the sgRNA sequence. In one embodiment, the sgRNA contains either an sgRNA recognition sequence or a full-length sgRNA sequence. The full-length sgRNA contains or consists of the sgRNA recognition sequence and a corresponding framework sequence, the framework sequence of which may contain a portion of the crRNA and the tracrRNA.

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

Furthermore, the sgRNA described in this invention can be modified, for example, by thiolation and/or methoxylation, to improve its stability. The sgRNA described in this invention can be synthesized by in vitro transcription or by chemical methods.

Methods for preparing modified CAR-T cells

This invention utilizes gene editing technology to knock out the TRAC molecule in CAR T cells, thereby preventing them from expressing the normal TCR molecule and reducing the transplant rejection effect of CAR T cells. In one aspect, this invention provides a method for preparing modified CAR-T cells, wherein the endogenous TRAC gene in the CAR-T cells is knocked out or inactivated. The modified CAR-T cells, due to the modification of the TCR molecule, exhibit reduced cellular immunogenicity and are considered universal CAR-T cells. Modified CAR-T cells are also referred to herein as TRAC ^KO CAR-T cells because they possess an inactivated, knocked-out, or very low-expressed TRAC gene.

In this invention, Cas9 cleaves the target locus in TRAC via sgRNA-guided cleavage, introducing DNA sequence insertion or base deletion to render the TRAC gene nonfunctional. Based on the disclosure of this application and common knowledge in the art, those skilled in the art will understand the various technical aspects regarding the selection and preparation of Cas9 protein and transfection methods.

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

In some embodiments, inactivating the endogenous TRAC gene includes introducing a complex with Cas nuclease and sgRNA into CAR-T cells. In some embodiments, inactivating the endogenous TRAC gene includes introducing sgRNA and Cas nuclease-encoding plasmids into CAR-T cells, respectively. The Cas nuclease includes Cas9 nuclease. Those skilled in the art will understand that the sgRNA of the present invention can be used in combination with various Cas proteins for use in various CRISPR/Cas systems, such as the CRISPR/Cas9 system, the CRISPR/nCas9 system, and the CRISPR/dCas9 system. The Cas9 protein is a multifunctional protein whose protein structure includes a recognition region (REC) composed of an α-helix, a nuclease region composed of an HNH domain and a RuvC domain, and a PAM binding region located at the C-terminus. These two important nuclease domains, RuvC and HNH, can respectively cleave the complementary and non-complementary strands of the gRNA DNA, producing blunt-ended DNA double-strand breaks. The Cas9 protein can be mutated as needed to form single-strand DNA breaks. In some embodiments, the Cas9 protein is wild-type Cas9. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes Cas9 protein or Staphylococcus aureus Cas9 protein. In some embodiments, the Cas9 protein induces double-strand breaks at target loci of the TRAC gene.

In the CRISPR/Cas9 gene editing system, sgRNAs play a crucial role in accurately recognizing target gene sequences. Their effectiveness influences editing efficiency and off-target effects, ultimately determining the success of gene editing. Therefore, designing appropriate and effective sgRNAs is fundamental to gene editing, and selecting suitable recognition sequences is the core task in sgRNA design. For designed sgRNAs, analysis can be conducted based on specificity scores, splicing efficiency scores, potential off-target scenarios, and off-target site information to select the optimal sgRNA.

Evaluation of the effects of TRAC gene editing

This invention also provides a method for evaluating the TRAC gene editing effect on modified CAR-T cells. In some embodiments, the method assesses the resistance of the modified CAR-T cells to heterologous immune cells (e.g., CAR-T cell viability after contact) by exposing the modified CAR-T cells to heterologous immune cells. In some embodiments, the immune cells are allogeneic to the CAR-T cells; for example, the immune cells include an MHC-I different from that of the CAR-T cells. In some embodiments, the immune cells are isolated from PBMCs of a subject different from the subject who received the CAR-T. 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 method identifies TRAC gene efficiency by combining a PCR reaction with T7E1 restriction enzyme digestion. The method includes extracting genomic DNA from modified CAR-T cells, designing primers for PCR amplification to obtain PCR products with knockout sites, adding T7E1 enzyme for digestion, and then performing agarose gel electrophoresis on the reaction products to determine the editing effect by the presence or absence of bands.

The α-constant region, encoded by the T-cell receptor α-constant region gene, is essential for the assembly of the endogenous TCR complex on the cell surface. Therefore, the use of sgRNAs targeting the T-cell receptor α-constant region gene as described herein results in reduced and/or knocked-out expression of T-cell receptors on the cell surface. In one aspect, the sgRNAs targeting the T-cell receptor α-constant region gene as described herein enhance the efficiency of modification of the human TCR α-constant region gene, for example, by reducing the expression of endogenous T-cell receptors (e.g., α/β T-cell receptors) on the cell surface of genetically modified CAR T cells by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100% compared to control cells.

This invention also provides a method for evaluating the tumor-killing effect of modified CAR-T cells. The method assesses the killing power of modified CAR-T cells against tumor cells by incubating modified CAR-T cells with tumor cells at a specific ratio. In some embodiments, the tumor cells are liver cancer cells, breast cancer cells, kidney cancer cells, lung cancer cells, etc.

This invention also provides a method for assessing the off-target probability of designed sgRNAs using Guide-seq. Guide-seq is a commonly used extracellular detection method for assessing off-target effects. It utilizes the NHEJ DNA repair mechanism, ligating a short double-stranded nucleotide sequence (dsODN) to the CRISPR-induced DNA double-strand break, essentially ligating the first-round adapter. Then, the genome is normally broken, and a second-round adapter is ligated on the other side. Libraries constructed in this way can obtain sequences on the off-target side; the easier it is to ligate a dsODN site, the higher the probability of off-target cleavage.

Systems, reagents, and kits for gene editing

In one aspect, this article provides modified CAR-T cells prepared by any of the methods provided herein. Specifically, an improved universal CAR-T cell is provided, wherein the TRAC gene is not expressed or is expressed at low levels. The universal CAR-T cells provided by this invention can effectively reduce the risk of graft-versus-host disease and immune rejection, thereby improving the therapeutic effect of CAR-T cells.

In one respect, this article provides pharmaceutical compositions comprising any of the modified CAR-T cells and pharmaceutically acceptable carriers provided herein.

In one respect, this article provides a composition comprising at least one sgRNA and a nuclease or an mRNA encoding a nuclease.

In one aspect, this document provides a gene editing system for inactivating endogenous TRAC genes in cells. In some embodiments, the system includes a nuclease or nucleic acid encoding the nuclease capable of cleaving a target site in an endogenous TRAC gene within the cellular genome. In some embodiments, the system includes an sgRNA having a recognition sequence complementary to a target sequence in the TRAC gene, the sgRNA being adapted to inactivate the endogenous TRAC gene. In some embodiments, the sgRNA includes a target sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of any one of SEQ ID NO: 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 present invention is particularly suitable for gene editing in CAR-T cells. In some embodiments, the gene editing system of the present invention is used to knock out the TRAC gene in CAR-T cells.

Exemplary implementation:

1. One or more sgRNAs comprising a recognition sequence for a target site in the TRAC gene, said recognition sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO: 1-27.

2. The sgRNA according to embodiment 1, wherein the recognition sequence has or consists of a nucleotide sequence of any one of SEQ ID NO: 1-27.

3. The sgRNA according to embodiment 1, wherein the sgRNA further comprises a frame sequence, wherein the frame sequence is preferably the sequence shown in SEQ ID NO:28.

4. The sgRNA according to any one of embodiments 1-3, wherein the sgRNA comprises or consists of the sequence shown in any one of SEQ ID NO:29-55.

5. A system for modifying the expression of the endogenous T cell receptor α homeostasis (TRAC) gene in cells, comprising:

The sgRNA or vector for expressing the sgRNA as described in any one of embodiments 1-4, and optionally, a nuclease or 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 and the vector for expressing sgRNA are separate vectors or the same vector.

9. The system according to any one of embodiments 5-8, wherein the cell is a cell capable of being used for allogeneic cell therapy, optionally, the cell is a T cell, such as a CAR-T cell.

10. A method for preparing TRAC gene-inactivated cells, the method comprising: contacting the cells with sgRNA as described in any one of embodiments 1-4 or a system as described in any one of embodiments 5-9, and introducing the sgRNA or the system into the cells, optionally inactivating the endogenous TRAC gene in the cells.

11. The method described in Implementation Scheme 10, the method further comprising the following steps:

1) Obtain cells, 2) Modify the cells to encode a chimeric antigen receptor against the target antigen, and 3) Edit the TRAC gene using the sgRNA of any one of embodiments 1-4 or the system of any one of embodiments 5-9, thereby preparing cells with inactivated TRAC gene.

The order of steps 2) and 3) can be interchanged, or steps 2) and 3) can be performed simultaneously.

12. The method of embodiment 10, wherein the nuclease in the system is made to form a complex with the sgRNA prior to contact.

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 according to any one of embodiments 10 to 13, wherein the cell is a cell that can be used for allogeneic cell therapy, such as T cells, preferably CAR-T cells.

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

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

17. A vector comprising a nucleotide sequence encoding an sgRNA of any one of embodiments 1-4, optionally said vector being a DNA vector such as a plasmid or a viral vector such as a retrovirus, adeno-associated virus, and lentivirus.

18. A reagent kit comprising:

A first container, comprising the sgRNA or an expression vector for expressing the sgRNA as described in any one of embodiments 1-4.

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

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

21. Use of the cells described in Embodiment 15 in the preparation of a medicament for treating cancer, autoimmune disease, or inflammatory disease in an allogeneic subject.

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

Beneficial effects of the present invention

1) The sgRNA provided by this invention for preparing universal CAR-T cells has high knockdown efficiency, good specificity, is not easy to be off-target, stably targets TRAC, has high cleavage efficiency, and greatly improves gene knockout efficiency.

2) The universal CAR-T cells provided by this invention can effectively reduce the risk of graft-versus-host disease and immune rejection, thereby improving the therapeutic effect of CAR-T cells.

Example

The following embodiments are provided to better understand the invention, and are not intended to be limiting.

Instruments and reagents

Example 1: T cell sorting experiment

Remove a cryovial containing 5 × ^10⁷ PBMC cells from the liquid nitrogen container and thaw it in a 37°C water bath. Place a new sterile cryovial in a magnetic rack and add 1 ml of X-VIVO medium. Then, take a 15 ml centrifuge tube, add 4 ml of X-VIVO medium, transfer the thawed PBMC cells to the 15 ml centrifuge tube, centrifuge (500 × g, 5 min), discard the supernatant, add 50 μl of Stemcell easysep separation antibody and 800 μl of X-VIVO medium, incubate for 5 min, add 50 μl of magnetic beads, resuspend the cells, and then transfer them to the prepared cryovial tube. Mix well and incubate for 3 min.

After incubation, take a 15ml centrifuge tube, add 7ml of culture medium, and transfer the cells from the cryopreservation tube to the centrifuge tube. Centrifuge at 500×g for 5min and count the cells. After counting, centrifuge again (500×g, 5min), discard the supernatant, and add 100μl of activating magnetic beads and 50μl of culture medium per 1× ^10⁷ cells, based on the count results. Resuspend the cells several times by pipetting, and incubate in an incubator for 7min. Repeat the incubation three times. After incubation, culture the cells in a T25 flask at a ratio of 2.5× ^10⁶ cells/ml of culture medium.

The T cells cultured overnight should be used promptly for lentivirus transfection the next day to construct CAR-T cells.

Example 2: Construction of CAR-T cells

293T cells intended for virus packaging were cultured in a cell culture incubator at 37°C and 5% _CO2 . The culture medium used was DMEM containing 10% Gibco fetal bovine serum. The day before the actual virus packaging, the cultured 293T cells were passaged in T75 cell flasks at a rate of 1 × ^10⁷ cells. Lentiviral packaging began when the 293T cells reached 70-80% confluence and were evenly distributed in the culture flasks.

Prepare a 1.5ml sterile centrifuge tube, add 460μl of serum-free DMEM medium, then add 40μl of PEI transfection reagent, mix thoroughly, and incubate for 5 min. Prepare another 1.5ml sterile centrifuge tube, add 15μl of CAR plasmid (Transfer Plasmid is based on the pCDH-CMV (addgene: 72265) plasmid, with the CMV promoter replaced by the EF1a promoter via software), then add 5μg of pMDLg/pRRE plasmid, 5μg of pMD2.G plasmid, and 5μg of pRSV-Rev plasmid, followed by 470μl of serum-free DMEM medium, and mix thoroughly. Add the medium containing PEI transfection reagent to the medium containing the plasmid to form the transfection system, mix thoroughly, and incubate for 15 min. Remove the 293T cells cultured overnight, discard the culture medium in the flask, carefully add 9 ml of serum-free DMEM medium, and then add 1 ml of transfection system. Return the cells to the incubator and continue culturing for 6 hours. After 6 hours, remove the culture flask, discard the culture medium, and carefully add 15 ml of serum-free DMEM medium. After 42 hours, collect the virus solution from the culture flask, and add another 15 ml of serum-free DMEM medium. After 24 hours, collect the virus solution from the culture flask again, and pour the total 30 ml of virus solution collected from both collections into a 50 ml sterile syringe. Filter the solution through a 0.45 μm filter membrane into a sterile ultracentrifuge tube. Centrifuge the tube at 21,000 × G for 2 hours, discard the supernatant, resuspend the virus pellet in 200 μl of X-VIVO medium, and store overnight at 4°C.

Remove 1× ^10⁶ T cells isolated the previous day from the incubator, add 100 μl of virus resuspended solution, and incubate at 37°C for 24 hours. After 24 hours, aspirate all cells, change the medium, and continue culturing. The cultured cells are the desired CAR-T cells.

Example 3: Electrical knockout experiment

Preparation of electroporation buffer: Add all of the replenishing solution to the dissolving solution, with a dissolving solution to replenishing solution ratio of 4.5:1. Prepare an appropriate amount of culture medium, place it in a well plate, and preheat it in an incubator. Dissolve TRAC-sgRNA to a solution of 100 pmol/μl. The TRAC-sgRNA includes 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. As shown in Table 1 below.

Table 1: TRAC-sgRNA recognition sequences

In this application, each full-length sgRNA sequence is equal to the recognition region sequence of the corresponding sgRNA plus the frame sequence shown in SEQ ID NO:28 (from the 5' end to the 3' end). That is, each full-length sgRNA sequence from the 5' end to the 3' end is composed of the recognition region sequence of the corresponding sgRNA and the frame sequence shown in SEQ ID NO:28.

TRAC-sgRNA was mixed with 24 pmol of Cas9 protein at a concentration of 72 pmol. The mixture was incubated for 10 min. After centrifugation and cell counting, 1 × ^10⁶ cells were centrifuged again and resuspended in 20 μl of electroporation buffer.

Add 20 μl of resuspended cell solution to each RNP. Transfer the cell solution into the strips. Turn on the electroporator and select the strip option. Use the T cell editing program CM119 to perform electroporation knockout. Select the wells where cell solution has been added and select the T cell editing option. Press the start button. After electroporation, transfer the mixture into the prepared culture medium and incubate in an incubator.

After electroporation and knockout, the cells were centrifuged and resuspended in 100 μl of PBS. 5 μl of APC Anti-CD3 antibody was added, and the cells were incubated at 4°C for 30 min. After centrifugation and discarding the supernatant, the cells were resuspended in 200 μl of PBS and analyzed by flow cytometry to determine the CD3 knockout efficiency, thereby evaluating the T cell receptor knockout efficiency. As shown in Figure 1, some of the screened sgRNAs can yield CAR-T cells with high TRAC knockout rates.

Example 4: GVHD Experiment

Remove a cryopreservation tube containing 5 × ^10⁷ PBMC cells from the liquid nitrogen tank and thaw it in a 37°C water bath. Take a clean 15ml centrifuge tube, add 9ml of culture medium, transfer the thawed cells to the centrifuge tube, centrifuge at 500×g for 5 minutes, discard the supernatant, add 10ml of culture medium, and mix thoroughly by pipetting.

Cell suspension was mixed with trypan blue at a 1:1 ratio, and cell concentration was measured using a cell counter. A sufficient amount of PBMC cells was aspirated, centrifuged (500×g, 5 min), the supernatant was discarded, and the cells were resuspended in 25 ml of culture medium and transferred to a T75 cell culture flask. PBMC cells were irradiated with 20 Gy units using an Elekta Infinity linear accelerator to induce cell death or pseudo-death. One × ^10⁶ irradiated PBMCs were used as target cells, and one × ^10⁶ CAR-T cells were used as effector cells and co-incubated with the PBMCs. After 12 hours, the exhaustion and activation of CAR-T cells were detected by flow cytometry.

As shown in Figure 2, flow cytometry analysis showed that the prepared TRAC ko CAR-T cells (with ko-TRAC1 and ko-TRAC13 as examples) had lower responsiveness to allogeneic T cells compared to mock CAR-T cells.

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

Preheat X-VIVO serum-free cell culture medium in a 37°C water bath. Prepare healthy 7860-luc, U251-luc, and Huh7-luc cells. Before plating for cell death, transfer the culture medium from the cultured 7860-luc, U251-luc, and Huh7-luc cells to a 15ml centrifuge tube, rinse the bottom of the culture flask with PBS, add an appropriate amount of 0.25% trypsin for digestion, and once the cells are suspended, pipette the original cell culture supernatant to the culture flask to stop digestion. After the cells are dispersed, transfer them to a 15ml centrifuge tube and centrifuge (400×g, 5min) to discard the supernatant. In a 96-well cell culture plate, add 60μl of 7860-luc, U251-luc, and Huh7-luc cells at a concentration of 3.33× ^10⁵ cells/ml to each well, resulting in 20,000 target cells per well. Place the cell culture plate containing the target cells in a 37°C, 5% CO2 incubator for 3-5 hours.

The CAR-T cells to be tested were adjusted in suspension concentration according to different positivity rates and effector-to-target ratios (E:T). With 20,000 target cells and an effector-to-target ratio of 8:1, and since the amount of culture medium added was also 60 μl, the cell concentration needed to be adjusted to (160,000/0.06/positivity rate) cells/ml. Simultaneously, effector-to-target ratios of 1:1, 1:2, and 1:4 were achieved by successively halving the concentration (150 μl cell suspension + 150 μl X-VIVO serum-free cell culture medium containing 10% FBS).

Incubate the cell culture plates with the cells at 37°C in a 5% CO2 incubator for 8 hours. Before the end of the incubation, remove the reagents from the ONE-Glo Luciferase Assay System kit from the -20°C freezer and allow them to thaw at room temperature. Dissolve the E606A powder in E605A reagent according to the instructions. After complete dissolution, aliquot the solution into EP tubes and store them at -20°C.

Turn on the multi-functional microplate reader and software, select Luminescence mode, and set up the plate layout. Add 100 μl of the prepared reagent to each well of cells, mix well by pipetting, and incubate at room temperature in the dark for 10 min. Use a pipette to transfer 180 μl of the solution from the cell culture plate into a 96-well white flat-bottomed plate, avoiding air bubbles. Place the 96-well white flat-bottomed plate in the microplate reader to read the data, and export and save the data for calculating the cell killing rate. Cell killing rate = (background luminescence value - sample luminescence value) / background luminescence value * 100%.

As shown in Figure 3, the knockout CAR-T cells have similar killing ability to the mock CAR-T cells and do not lose their killing ability due to knockout. At the same time, they also do not have the ability to kill non-target cells, which proves that the prepared knockout CAR-T cells have good specific tumor killing ability.

Example 6: T7E1 enzyme digestion experiment

Prepare 5 × ^10⁶ TRAC ^KO CAR-T cells, centrifuge at 250 × g for 5 min, discard the supernatant, and resuspend the cells in 200 μl PBS. Cell genome extraction was performed using... Genomic DNA kit. Add 20 μl Proteinase K to the cells, followed by 20 μl RNase A. Vortex briefly to mix, then incubate at room temperature for 2 min. Add 200 μl... After mixing the genome lysis/binding buffer by vortexing again, incubate at 55°C for 10 min. Add 200 μl of anhydrous ethanol to the lysis buffer and stir for 5 seconds to form a homogeneous solution.

Add the homogeneous solution to Centrifuge the separation column at 10000×g for 1 min. After centrifugation, remove the separation column and place it in a clean container. Add 500 μl of Wash Buffer 1 (prepared with ethanol) to the column and centrifuge at 10000g for 1 min. After centrifugation, remove the column and place it in a clean container. In a collection tube, add 500 μl of Wash Buffer 2 prepared with ethanol to the column and centrifuge at 20000g for 3 min. Place the separation column in a sterile 1.5 ml centrifuge tube and add 100 μl of [unspecified ingredient] to the column. Genome elution buffer. Incubate at room temperature for 1 min, then centrifuge at 20,000 rpm for 1 min. Take a new sterile 1.5 ml centrifuge tube, place the separation column inside, and add 100 μl of [unspecified ingredient] to the separation column. Genome elution buffer. Incubate at room temperature for 1 min, then centrifuge at 20000g for 1.5 min.

The DNA concentration was quantified using a micro-ultraviolet spectrophotometer. After adjusting the DNA concentration, 200 ng of DNA was extracted and PCR amplified using high-fidelity DNA polymerase to obtain PCR products with knockout sites. The primers used in one example experiment are shown in Table 2.

Table 2. TRAC amplification primer sequences

Similarly, perform all the above operations on the non-knockout CAR-T cells to obtain the PCR product of the non-knockout group. Purify the PCR product using the Beyotime DNA Purification Kit. Add an equal volume of DNA purification binding buffer to the PCR product and mix well. Add the homogeneous solution to the DNA purification column, incubate for 1 min, and then centrifuge at 20000×g for 5 min. After centrifugation, discard the liquid in the collection tube. Add 700 μl of washing buffer to the DNA purification column, incubate for 1 min, and then centrifuge at 20000×g for 1 min. After centrifugation, discard the liquid in the collection tube. Add another 500 μl of washing buffer to the DNA purification column and centrifuge at 20000×g for 1 min. Further wash away impurities and discard the liquid in the collection tube. Centrifuge again to remove the remaining liquid and allow residual ethanol to evaporate completely. Place the DNA purification column on a 1.5 ml centrifuge tube and add 50 μl of elution buffer to the center of the column to allow the liquid to be absorbed by the purification column, and let stand for 1 min. After centrifugation at 20000×g for 1 min, the resulting liquid is high-purity DNA. The Novozymes T7 Endonuclease I kit was used for the T7E1 experiment. The T7E1 enzyme digestion reaction system is shown in Table 3.

Table 3. T7E1 enzyme digestion reaction system

After preparing the T7E1 enzyme digestion reaction system, an annealing reaction was carried out. The annealing procedure is shown in Table 4.

Table 4. Annealing procedure for T7E1 enzyme digestion reaction

Add 1 μl of T7E1 to the annealing product and incubate at 37°C for 30 min. Terminate the digestion reaction by adding 1.5 μl of 0.25 M EDTA. Detect the digestion product directly by 2% agarose gel electrophoresis.

As shown in Figure 4, gel imaging analysis revealed that some of the screened sgRNAs could achieve high TRAC knockout efficiency.

Example 7: Guide-seq

Cell culture and molecular knife transfection: Cells were cultured and transfected with CRISPR/Cas9 gene scissors and dsODN tags. A dsODN tag is a DNA fragment containing primers and barcode sequences used to label the DNA sequence cleaved by Cas9. The purpose of this step is to guide the Cas9 protein to target specific DNA sequences within the cell and to label the cleaved DNA sequences.

DNA extraction: Genomic DNA is extracted from cells. The purpose of this step is to obtain the DNA sequence cut by Cas9 and to perform subsequent library construction.

PCR amplification: PCR amplification was performed using forward and reverse ODN primers. dsODN tag primers were ligated to the DNA sequence cleaved by Cas9, and a sufficient number of DNA fragments were amplified for subsequent sequencing analysis.

Next-generation sequencing: High-throughput sequencing is performed on the DNA fragments amplified by PCR. By determining the barcode sequence and corresponding target sequence of each primer, the target location of the Cas9 primers can be determined. After sequencing, the data is further analyzed.

Sample splitting: Since barcode sequences are used during GUIDE-seq library construction, the sequencing reads of each sample need to be split according to their barcode sequences. Therefore, the sequencing data of each sample needs to be separated first for subsequent analysis.

Removing PCR duplications: During PCR amplification, the same DNA fragment may be amplified multiple times, leading to PCR duplication. To avoid this effect, sequencing data needs to be deduplicated to reduce the bias introduced by PCR amplification and improve the accuracy and reliability of data analysis.

Alignment: The deduplication-removed sequencing data is aligned with the reference genome to determine the target sequence of each primer, thereby identifying the DNA sequence cut by Cas9 and accurately determining its target location.

Identifying candidate sites and off-target sequences: Based on the alignment results, candidate sites and off-target sequences can be identified. Candidate sites refer to the target sequences that are cleaved by Cas9, while off-target sequences are DNA sequences similar to the target sequences but not cleaved by Cas9. The purpose of this step is to evaluate the specificity and accuracy of the CRISPR system and to identify possible site mutations or insertions/deletions.

The report will sort the identified sites by read count and annotate them. It will also summarize and report the analysis results, describing information about Cas9 target sites and off-target sequences.

Visualization: The detected on-target and off-target sites are visualized using software such as IGV to provide intuitive results and facilitate better understanding of the analysis.

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

Although exemplary embodiments of the present invention have been shown and described in this application, those skilled in the art will understand that the above embodiments should not be construed as limiting the scope of the present invention, and that changes, substitutions and modifications may be made without departing from the spirit, principles and scope of the present invention.

Claims (22)

One or more sgRNAs comprising a recognition sequence for a target site in the TRAC gene, said recognition sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO:1-27. The sgRNA of claim 1, wherein the recognition sequence has or is composed of any of the nucleotide sequences in SEQ ID NO: 1-27. The sgRNA of claim 1, wherein the sgRNA further comprises a frame sequence, preferably the sequence shown in SEQ ID NO:28. The sgRNA of any one of claims 1-3, wherein the sgRNA comprises or consists of the sequence shown in any one of SEQ ID NO:29-55. A system for modifying the expression of the endogenous T cell receptor α constant region (TRAC) gene in cells, comprising: The sgRNA or vector for expressing the sgRNA according to any one of claims 1-4, and optionally, a nuclease or a nucleic acid encoding the nuclease. The system of claim 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. The system of claim 6, wherein the nuclease forms a complex with the sgRNA. The system of claim 5 or 6, wherein the system comprises a vector encoding the nuclease, optionally, the vector encoding the nuclease and the vector for expressing sgRNA are separate vectors or the same vector. The system of any one of claims 5-8, wherein the cell is a cell capable of being used for allogeneic cell therapy, optionally, the cell is a T cell, such as a CAR-T cell. A method for preparing cells with inactivated TRAC genes, the method comprising: contacting the cells with sgRNA of any one of claims 1-4 or a system of any one of claims 5-9, and introducing the sgRNA or the system into the cells, optionally inactivating endogenous TRAC genes in the cells. The method of claim 10, further comprising the following steps: 1) Obtaining cells, 2) Modifying the cells to encode a chimeric antigen receptor against the target antigen, and 3) Editing the TRAC gene using the sgRNA of any one of claims 1-4 or the system of any one of claims 5-9, thereby preparing cells with inactivated TRAC gene. The order of steps 2) and 3) can be interchanged, or steps 2) and 3) can be performed simultaneously. The method of claim 10, wherein the nuclease in the system is made to form a complex with the sgRNA prior to contact. The method of claim 10 or 11, wherein the method further comprises introducing the sgRNA or system into the cell by electrotransfection. 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 T cells, preferably CAR-T cells. A cell prepared by any one of claims 10 to 14. A nucleic acid molecule encoding the sgRNA of any one of claims 1-4. A vector comprising a nucleotide sequence encoding an sgRNA of any one of claims 1-4, optionally said vector being a DNA vector such as a plasmid or a viral vector such as a retrovirus, adeno-associated virus, or lentivirus. A reagent kit comprising: A first container, comprising the sgRNA of any one of claims 1-4 or an expression vector for expressing the sgRNA. The kit of claim 18 further comprises a second container containing a nuclease or a nucleic acid encoding the nuclease. The kit of claim 18, wherein the vector for expressing sgRNA further comprises a nucleic acid sequence encoding a nuclease. The use of the cells of claim 15 in the preparation of a medicament for treating cancer, autoimmune disease, or inflammatory disease in an allogeneic subject. Use of the sgRNA of any one of claims 1-4 or the system of any one of claims 5-9 in the preparation of T cells with inactivated endogenous T cell receptor α constant region (TRAC) gene, wherein the T cells are preferably CAR-T cells.