Gene editing systems and methods for reducing immunogenicity and graft versus host response

Abstract

The present application provides gene editing systems and methods for reducing cellular immunogenicity and reducing graft versus host (GvH) response or promoting persistence of therapeutic cells. Also provided are polynucleotides, vectors, cells, kits, and compositions comprising components of the gene editing system, as well as methods related to treatments for reducing cellular immunogenicity and reducing GvH response.

Description

Technical Field

[0001] The present disclosure generally relates to gene editing systems and methods for reducing immunogenicity and / or reducing graft-versus-host (GvH) reactions, and / or promoting the persistence of therapeutic cells. Also disclosed are polynucleotides, vectors, cells, kits, and compositions comprising components of the gene editing system, as well as methods related to therapies for reducing immunogenicity and / or reducing GvH reactions and / or promoting the persistence of therapeutic cells.

[0002] Cross-reference to Related Applications

[0003] This application claims the priority and benefits of International Application No. PCT / CN2022 / 128215 filed on October 28, 2022, and International Application No. PCT / CN2023 / 080709 filed on March 10, 2023, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing submitted electronically in the form of an XML file named "sequencelisting.xml", which is 1,053,655 bytes in size and was created on October 27, 2023. The information contained in the sequence listing is incorporated herein by reference. Background Art

[0006] Chimeric antigen receptor (CAR)-T cell therapy, as a promising cancer immunotherapy, has revolutionized anti-tumor treatment, especially for hematological malignancies, by producing significant long-term anti-tumor effects and having higher target specificity. The US Food and Drug Administration (FDA), the European Medicines Agency (EMA), or the National Medical Products Administration of China (NMPA) have approved several autologous CAR-T cell therapy products, mainly for the treatment of refractory or relapsed lymphoma or myeloma. (Ghaffari, Sasan, Nastaran Khalili, and Nima Rezaei. "CRISPR / Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy." Journal of Experimental & Clinical Cancer Research 40.1 (2021): 269.)

[0007] However, conventional autologous CAR-T cell therapy has some limitations, such as long manufacturing cycles, manufacturing failures in some patients, and high costs, which have cast a shadow on the development of autologous CAR-T cell therapy. Therefore, the development of universal / allogeneic CAR-T (UCAR-T) or enhanced autologous CAR-T cell therapy is an attractive breakthrough point that can overcome some of these drawbacks.

[0008] Despite its advantages compared to autologous CAR-T therapy, allogeneic CAR-T cell therapy has two major challenges. First, the administered allogeneic T cells can cause life-threatening graft-versus-host disease (GvHD). Second, allogeneic T cells may be rapidly cleared by the host immune system, limiting the persistence of their anti-tumor activity.

[0009] GvHD is one of the main causes of death after allogeneic hematopoietic stem cell transplantation and thus must be prevented. Since the alloreactivity of T cells depends on the interaction of the T cell receptor (TCR) with allogeneic antigens presented by human leukocyte antigen (HLA), TCR-depleted T cells do not cause GvH reactions when infused into HLA-mismatched patients. Some studies have attempted to reduce the risk of GvHD by using small interfering RNA, ZFN, TALEN, megaTAL nucleases, engineered homing endonucleases, or CRISPR / Cas9 to genetically ablate the TCR locus (primarily the TCRα constant region (TRAC)). On the other hand, to prevent or reduce host immune rejection, one approach is to reduce the immunogenicity of allogeneic T cells, for example, by genetically abolishing the β2-microglobulin (B2M) gene to disrupt MHC class I molecules. Another approach is to delete CD52 from donor T cells and use anti-CD52 monoclonal antibodies to eliminate host T cells (which express CD52) to avoid allogeneic rejection. In addition, inhibitory checkpoints (e.g., PD-1 encoded by the PDCD1 gene, CTLA4 gene, TIGIT gene, LAG3 gene, and TIM-3 gene) can be knocked out individually or simultaneously with each other, and / or with TRAC, CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and / or CD38 individually or simultaneously, to enhance the potency and persistence of autologous or allogeneic CAR-T cells or NK cells. For anti-CD7 or anti-CD38 CAR-T cells, CD7 or CD38 can be knocked out to prevent fratricide. The FAS / FASL signal induces apoptosis of cytotoxic T cells, which weakens the antitumor efficacy of CAR-T therapy. TGF-β secreted in the tumor microenvironment (TME) inhibits T cell function by binding to TGFBR2. The cytokine-induced SH2-containing (CISH) protein is induced in CD8+ T cells upon TCR stimulation and inhibits T cell antitumor function. CISH is also a key negative regulator of IL-15 signaling in NK cells. CBLB has been recognized as an intracellular checkpoint in T cells and NK cells, and the deletion of CBLB enhances the function of T cells and NK cells. The KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is an effective NK cell immune checkpoint.

[0010] While most groups use nucleases (ZNF, TALEN, or CRISPR / Cas9) to knock out these targets, there are reports regarding the risks of these modifications as they all result in double-strand breaks (DSBs), which will activate the p53 pathway, affect cell growth, and induce chromosomal translocations. Duplex or multiplex editing will further increase this risk. Therefore, the need to develop safer methods for modifying these targets remains unmet.

[0011] The combination of CRISPR-Cas9 and cytidine deaminases (APOBEC / AID) generates cytosine base editors (CBEs) for programmable cytosine-to-thymine (C-to-T) substitution. Such CBEs have been applied to achieve efficient editing in various species successfully. Since this base editing process does not rely on the generation of DNA double-strand breaks (DSBs), unwanted nucleotide insertions / deletions (indels) or DNA damage responses (DDRs) can be largely avoided.

[0012] The safety and efficiency of gene editing tools are crucial in clinical applications. Although CBEs do not cause DSBs or activate the p53-mediated DDR pathway as Cas9 nucleases do, APOBEC / AID family members can trigger C-to-T base substitutions in single-stranded DNA (ssDNA) regions, which are randomly formed during various cellular processes, including DNA replication, repair, and transcription. Therefore, the specificity of previous base editing systems is affected, which may limit the application of CBEs for therapeutic purposes. Summary of the Invention

[0013] The present disclosure provides gene editing systems, polynucleotides, vectors, cells, compositions, kits, and methods for reducing immunogenicity and graft-versus-host reactions. In some embodiments, the present disclosure provides gene editing systems that target genes selected from TRAC, CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0014] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the T cell receptor α constant (TRAC) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 1-5.

[0015] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 2. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 26.

[0016] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD52 gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 6-8.

[0017] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 3. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 27.

[0018] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the beta-2 microglobulin (B2M) gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 9-19.

[0019] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 4. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 28.

[0020] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the programmed cell death protein 1 (PDCD1) gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 20-38.

[0021] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 5. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 29.

[0022] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytotoxic T-lymphocyte associated protein 4 (CTLA4) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 247-256.

[0023] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 13.

[0024] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the T cell immunoreceptor with Ig and ITIM domains (TIGIT) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 278-294.

[0025] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 14.

[0026] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the hepatitis A virus cellular receptor 2 (HAVCR2 / TIM3) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 323-337.

[0027] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 15.

[0028] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the lymphocyte activation 3 (LAG3) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 364 - 396.

[0029] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 16.

[0030] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytokine-inducible SH2-containing protein (CISH) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 472 - 482.

[0031] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 17. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 30.

[0032] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the transforming growth factor beta receptor 2 (TGFBR2) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 504 - 510.

[0033] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 18. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 31.

[0034] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the Fas cell surface death receptor (FAS) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 530-541.

[0035] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 19. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 32.

[0036] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD7 gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 565-575.

[0037] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 20.

[0038] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the Cbl proto-oncogene B (CBLB) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 609-618.

[0039] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 21. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 33.

[0040] In one aspect, the present disclosure provides a gene editing system comprising a master guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the killer cell lectin-like receptor C1 (KLRC1) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 637-641.

[0041] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 22. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 34.

[0042] In one aspect, the present disclosure provides a gene editing system comprising a master guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD38 gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 651-659.

[0043] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences listed in Table 23. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise the sequences listed in Table 35.

[0044] In some embodiments, the gene editing system disclosed herein comprises (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, and (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, and wherein the first Cas protein and the second Cas protein are the same or different.

[0045] In some embodiments, the gene editing systems disclosed herein comprise (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, and (6) a protease, or a polynucleotide encoding the protease, and (7) a nucleobase deaminase inhibitor domain, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked by a linker to the nucleobase deaminase or its catalytic domain in the first fusion protein, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain.

[0046] In some embodiments, the gene editing systems disclosed herein comprise (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising the protease and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked by a linker to the nucleobase deaminase or its catalytic domain in the first fusion protein, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the protease and the second RNA-binding domain are optionally linked by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA-binding domain binds to the second protein-binding motif.

[0047] In some embodiments, the protease is split into a first protease fragment and a second protease fragment, wherein neither the first nor the second protease fragment alone can cleave the cleavage site.

[0048] In some embodiments, the gene editing systems disclosed herein comprise (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, and (9) a third fusion protein comprising a second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA-binding domain binds to the second protein-binding motif, and wherein the third RNA-binding domain binds to the third protein-binding motif.

[0049] In some embodiments, the gene editing systems disclosed herein comprise (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, and (9) a third fusion protein comprising a second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA-binding domain binds to the second protein-binding motif, wherein the third RNA-binding domain binds to the third protein-binding motif, and wherein the second and third RNA-binding domains are the same or different, and the second and third protein-binding motifs are the same or different.

[0050] In some embodiments, the gene editing systems disclosed herein comprise (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA-binding domain binds to the second protein-binding motif.

[0051] In some embodiments, the protease is TEV protease, TuMV protease, PPV protease, PVY protease, ZIKV protease, or WNV protease.

[0052] In some embodiments, the protease is TEV protease. In some embodiments, the TEV protease comprises the sequence shown in SEQ ID NO:124.

[0053] In some embodiments, the first TEV protease fragment comprises the sequence of SEQ ID NO:125.

[0054] In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of the nucleobase deaminase.

[0055] In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of the cytidine deaminase.

[0056] In some embodiments, the inhibitory domain of the cytidine deaminase comprises the amino acid sequence shown in SEQ ID NO: 141 or SEQ ID NO: 142.

[0057] In some embodiments, the nucleotide deaminase is a cytidine deaminase.

[0058] In some embodiments, the cytidine deaminase is selected from APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), APOBECI (Al), APOBEC3 (A3), APOBEC2 (A2), APOBEC4 (A4), and AICDA (AID).

[0059] In some embodiments, the cytidine deaminase is a human or murine cytidine deaminase.

[0060] In some embodiments, the catalytic domain of the cytidine deaminase is murine A3 cytidine deaminase domain 1 (mA3 - CDAl) or human A3B cytidine deaminase domain 2 (hA3B - CDA2).

[0061] In some embodiments, the first fusion protein further comprises a uracil glycosylase inhibitor (UGI).

[0062] In some embodiments, the first fusion protein further comprises a nuclear localization sequence (NLS).

[0063] In some embodiments, the Cas protein is Cas9, dead Cas9 (dCas9), or Cas9 nickase (nCas9) selected from the following: SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl, LbCpfl, FnCpfl, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9 - NG, xSpCas9, RHA FnCas9, KKHSaCas9, NmeCas9, StCas9, CjCas9, AsCpfl, FnCpfl, SsCpfl, PcCpfl, BpCpfl, CmtCpfl, LiCpfl, PmCpfl, Pb3310Cpfl, Pb4417Cpfl, BsCpfl, EeCpfl, BhCasl2b, AkCasl2b, EbCasl2b, LsCasl2b, RfCasl3d, LwaCasl3a, PspCasl3b, PguCasl3b, and RanCasl3b.

[0064] In some embodiments, the Cas protein is nCas9. In some embodiments, the nCas9 protein is the nCas9-D10A protein. In some embodiments, the nCas9-D10A protein has the amino acid sequence of SEQ ID NO:146.

[0065] In some embodiments, the first protein-binding RNA motif and the first RNA-binding domain, the second protein-binding RNA motif and the second RNA-binding domain, and the third protein-binding RNA motif and the third RNA-binding domain are each independently selected from the MS2 bacteriophage operator stem-loop and the MS2 coat protein (MCP) or its RNA-binding segment; BoxB and N22P or its RNA-binding segment; the telomerase Ku-binding motif and the Ku protein or its RNA-binding segment; the telomerase Sm7-binding motif and the Sm7 protein or its RNA-binding segment; the PP7 bacteriophage operator stem-loop and the PP7 coat protein (PCP) or its RNA-binding segment; the SfMu bacteriophage Com stem-loop and the Com RNA-binding protein or its RNA-binding segment; and non-natural RNA aptamers and the corresponding aptamer ligands or their RNA-binding segments.

[0066] In another aspect, the present disclosure provides polynucleotides encoding the hgRNAs and / or mRNAs disclosed herein.

[0067] In another aspect, the present disclosure provides polynucleotides encoding all components of the gene editing system disclosed herein except the first and second Cas proteins.

[0068] In another aspect, the present disclosure provides a kit comprising polynucleotides encoding all components of the gene editing system disclosed herein except the first and second Cas proteins, and polynucleotides encoding the first and / or second Cas proteins of the gene editing system disclosed herein. In some embodiments, the first and second Cas proteins are the same Cas protein.

[0069] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding the hgRNAs and / or mRNAs disclosed herein.

[0070] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding all components of the gene editing system disclosed herein except the first and second Cas proteins.

[0071] In some embodiments, the vector is a plasmid or a viral vector.

[0072] In some embodiments, the vector is a polycistronic vector.

[0073] In another aspect, the present disclosure provides a kit comprising the vectors disclosed above, and a vector comprising a polynucleotide encoding the first and / or second Cas protein in the gene editing system disclosed herein.

[0074] In another aspect, the present disclosure provides a cell comprising any one or more of the gene editing systems disclosed herein.

[0075] In another aspect, the present disclosure provides a cell comprising the polynucleotides disclosed herein. In some embodiments, the cell further comprises a polynucleotide encoding the first and / or second Cas protein in the gene editing system disclosed herein.

[0076] In another aspect, the present disclosure provides a cell comprising the vectors disclosed herein. In some embodiments, the cell further comprises a vector comprising a polynucleotide encoding the first and / or second Cas protein in the gene editing system disclosed herein.

[0077] In another aspect, the present disclosure provides a cell comprising the components of the kit disclosed herein.

[0078] In some embodiments, the cell is a stem cell.

[0079] In some embodiments, the cell is a pluripotent stem cell or a hematopoietic stem cell.

[0080] In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or an embryonic stem cell.

[0081] In some embodiments, the cell is an immune cell.

[0082] In some embodiments, the cell is selected from T cells, B cells, natural killer cells (NK cells), macrophages, dendritic cells, monocytes, granulocytes, and mast cells.

[0083] In some embodiments, the cell is a T cell.

[0084] In some embodiments, the T cell comprises a chimeric antigen receptor (CAR).

[0085] In some embodiments, the T cell is a CAR-T cell.

[0086] In some embodiments, the cell is a natural killer cell (NK cell).

[0087] In some embodiments, the NK cell is a CAR-NK cell.

[0088] In some embodiments, the cell is a primary cell.

[0089] In some embodiments, the cells are differentiated cells.

[0090] In some embodiments, the cells are differentiated from pluripotent stem cells. In some embodiments, the cells are differentiated from iPSCs or ESCs.

[0091] In another aspect, the present disclosure provides a composition comprising any one or more of the gene editing systems disclosed herein.

[0092] In another aspect, the present disclosure provides a composition comprising the cells disclosed herein.

[0093] In another aspect, the present disclosure provides a kit comprising one or more of the gene editing systems disclosed herein. For example, the present disclosure provides a kit comprising a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting the TRAC gene, the B2M gene, and / or the CD52 gene. For example, in some embodiments, the present disclosure provides a kit comprising a first gene editing system and a second gene editing system, wherein the first gene editing system and the second gene editing system each target a gene selected from TRAC, CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0094] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TRAC gene and a second gene editing system targeting a gene selected from CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, and CD38.

[0095] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CD52 gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0096] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the B2M gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0097] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting a gene selected from CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0098] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CTLA4 gene and a second gene editing system targeting a gene selected from TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0099] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TIGIT gene and a second gene editing system targeting a gene selected from TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0100] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TIM3 gene and a second gene editing system targeting a gene selected from LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0101] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the LAG3 gene and a second gene editing system targeting a gene selected from CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0102] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CISH gene and a second gene editing system targeting a gene selected from TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0103] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TGFBR2 gene and a second gene editing system targeting a gene selected from FAS, CD7, CBLB, KLRC1, and CD38.

[0104] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the FAS gene and a second gene editing system targeting a gene selected from CD7, CBLB, KLRC1, and CD38.

[0105] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CD7 gene and a second gene editing system targeting a gene selected from CBLB and KLRC1.

[0106] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CBLB gene and a second gene editing system targeting a gene selected from KLRC1 and CD38.

[0107] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting CD38.

[0108] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting CD38.

[0109] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting a gene selected from PD1, TGFBR2, CISH, CD38, CBLB, TIGIT, TIM-3, LAG3, FAS and TGFBR2.

[0110] In another aspect, the present disclosure provides a method for reducing the immunogenicity of a cell, comprising introducing one or more of the gene editing systems disclosed herein into the cell.

[0111] In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell, B cell, natural killer cell (NK cell), macrophage, dendritic cell, monocyte, granulocyte or mast cell. In some embodiments, the immune cell comprises a chimeric antigen receptor. In some embodiments, the cell is a T cell. In some embodiments, the T cell comprises a chimeric antigen receptor. In some embodiments, the T cell is a CAR-T cell. In some embodiments, the cell is an NK cell. In some embodiments, the NK cell comprises a chimeric antigen receptor. In some embodiments, the NK cell is a CAR-NK cell. In some embodiments, the cell differentiates from a pluripotent stem cell. In some embodiments, the cell differentiates from an iPSC or ESC. In some embodiments, the cell is a primary cell.

[0112] In another aspect, the present disclosure provides a method for reducing graft-versus-host (GvH) reactions associated with administering allogeneic cells to a subject, comprising reducing the immunogenicity of allogeneic cells by introducing any one or more of the gene editing systems disclosed herein into the allogeneic cells.

[0113] In some embodiments, the allogeneic cells are immune cells. In some embodiments, the immune cells are T cells, B cells, natural killer cells (NK cells), macrophages, dendritic cells, monocytes, granulocytes, or mast cells. In some embodiments, the immune cells comprise chimeric antigen receptors. In some embodiments, the allogeneic cells are T cells. In some embodiments, the T cells comprise chimeric antigen receptors. In some embodiments, the T cells are CAR-T cells. In some embodiments, the allogeneic cells are NK cells. In some embodiments, the NK cells comprise chimeric antigen receptors. In some embodiments, the NK cells are CAR-NK cells. In some embodiments, the cells are differentiated from pluripotent stem cells. In some embodiments, the cells are differentiated from iPSCs or ESCs. In some embodiments, the cells are primary cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Exemplary base editors that can be used in the gene editing systems disclosed herein are illustrated. Various versions of the base editors are designated as V1, V2, V3, V4, and V5, and the constructs are designated as tBE-V1-rA1, tBE-V2-rA1, tBE-V3-rA1, tBE-V4-rA1, tBE-V5-rA1, and tBE-V5-mA3. Figure 1 A shows a schematic illustration demonstrating the construction and development of various versions of the base editors. Figure 1 B shows the interactions of the molecular components in different versions of the base editors. The base editors of V2 to V5 illustrate different strategies for cleaving mA3dCDI. The dCDI domain can be cleaved from APOBEC by a two-component interaction of the TEV site with free TEV protease (V2), N22p-fused TEV protease (V3), or TEV protease reconstituted by the mgRNA-boxB (V4). In version 5 (V5) of the base editor, dCDI is cleaved from APOBEC by a three-component interaction of the TEV site, TEVn, and N22p-TEVc.

[0115] Figure 2 Shows the editing efficiency induced by tBE with an mgRNA targeting human TRAC and its hgRNA pair. Figure 2A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human TRAC gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 2 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 2 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human TRAC gene calculated by EditR analysis.

[0116] Figure 3 is the validation of TRAC knockout (KO) at the protein level. Figure 3 A - C are the results of flow cytometry analysis of the surface CD3 levels of MOCK (A) or cells transfected with tBE + TRAC - mg2 - U1 (B) or cells transfected with tBE + TRAC - mg4 - U2 (C) in Jurkat T cells (CD3 forms the TCR - CD3 complex with TCR). Figure 3 D is Figure 3 a summary of the CD3+ cell ratios in A - C. Figure 3 E - G are the results of flow cytometry analysis of the surface CD3 levels of MOCK (E) or cells transfected with tBE + TRAC - mg2 - U1 (F) or cells transfected with tBE + TRAC - mg4 - U2 (G) in primary T cells. Figure 3 H is a summary of the CD3+ cell ratios in E - G.

[0117] Figure 4 represents the editing efficiency induced by tBE with the mgRNA and its hgRNA pair targeting human CD52. Figure 4 A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human CD52 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 4 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 4 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human CD52 gene calculated by EditR analysis.

[0118] Figure 5 is the validation of CD52 KO or TRAC / CD52 double KO at the protein level. Figure 5Flow cytometry analysis results of surface CD3 levels and CD52 protein levels in MOCK (A), cells transfected with tBE + TRAC-mg2-U1 + CD52-mg2-U3 (B), or cells transfected with tBE + TRAC-mg2-U1 + CD52-mg3-U2 (C) in Jurkat T cells. Figure 5 D is a summary of the CD3+ or CD52+ cell ratios in 5A - C. Figure 5 Flow cytometry analysis results of CD52 protein levels in MOCK (E) or cells transfected with tBE + CD52-mg2-U3 (F) in primary T cells. Figure 5 G is a summary of the CD52+ cell ratios in 5E - F. Figure 5 Flow cytometry analysis results of surface CD3 levels and CD52 protein levels in MOCK (H), cells transfected with tBE + TRAC-mg2-U1 + CD52-mg2-U3 (I), or cells transfected with tBE + TRAC-mg4-U2 + CD52-mg2-U3 (J) in primary T cells. Figure 5 K is a summary of the CD3+ or CD52+ cell ratios in 4H - J.

[0119] Figure 6 Indicates the editing efficiency induced by tBE and the mgRNA and its hgRNA targeting human B2M. Figure 6 A is a schematic diagram illustrating the co - transfection of mgRNA and its different hgRNAs targeting the human B2M gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 6 B shows the editing efficiency induced by tBE - V5 - mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 6 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human B2M gene calculated by EditR analysis.

[0120] Figure 7 Is the verification of B2M KO or TRAC / B2M double KO at the protein level. Figure 7 Flow cytometry analysis results of B2M protein levels in MOCK (A), cells transfected with tBE + B2M - mg1 - U3 (B), or cells transfected with tBE + B2M - mg2 - U1 (C) in Jurkat T cells. Figure 7 D is a summary of the B2M+ cell ratios in 7A - C. Figure 7E - G are the results of flow cytometry analysis of B2M and TRAC protein levels in primary T cells that were either MOCK (E), transfected with tBE + B2M - mg1 - U3 (F), or transfected with tBE + B2M - mg1 - U3+TRAC - mg4 - U2 (G). Figure 7 H is a summary of the TRAC+ or B2M+ cell ratios of 7E - G.

[0121] Figure 8 Indicates the editing efficiency induced by tBE with the mgRNA targeting human PDCD1 and its hgRNA pair. Figure 8 A is a schematic diagram illustrating the co - transfection of the mgRNA targeting the human PDCD1 gene and its different hgRNAs with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 8 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 8 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human PDCD1 gene calculated by EditR analysis.

[0122] Figure 9 Is the validation of PDCD1 KO or TRAC / CD52 / PDCD1 triple KO at the protein level. Figure 9 A - D are the results of flow cytometry analysis of PD1 protein levels in primary T cells that were either MOCK (A), transfected with tBE + PDCD1 - mg6 - U2 (B), transfected with tBE + PDCD1 - mg7 - U2 (C), or transfected with PDCD1 - mg15 - U1. Figure 9 E is a summary of the PD1+ cell ratios of 9A - D. Figure 9 F - M are the results of flow cytometry analysis of surface CD3 levels and CD52 protein levels (F - I) and PD1 protein levels (J - M) in primary T cells that were either MOCK (F,J), transfected with tBE + TRAC - mg4 - U2 (G,K), transfected with tBE + TRAC - mg4 - U2+CD52 - mg2 - U3 (H,L), or transfected with tBE + TRAC - mg4 - U2+CD52 - mg2 - U3+PDCD1 - mg7 - U2 (I,M). Figure 9 N is a summary of the CD3+, CD52+ or PD1+ cell ratios of 9F - M.

[0123] Figure 10 Indicates the editing efficiency induced by tBE with the mgRNA targeting human CTLA4 and its hgRNA pair. Figure 10A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human CTLA4 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 10 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 10 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human CTLA4 gene calculated by EditR analysis.

[0124] Figure 11 Represents the editing efficiency induced by tBE with mgRNAs and their hgRNA pairs targeting human TIGIT. Figure 11 A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human TIGIT gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 11 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 11 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human TIGIT gene calculated by EditR analysis.

[0125] Figure 12 Represents the editing efficiency induced by tBE with mgRNAs and their hgRNA pairs targeting human TIM3. Figure 12 A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human TIM3 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 12 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 12 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human TIM3 gene calculated by EditR analysis.

[0126] Figure 13 Represents the editing efficiency induced by tBE with mgRNAs and their hgRNA pairs targeting human LAG3. Figure 13 A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human LAG3 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 13Panel B shows the editing efficiency induced by tBE-V5-mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 13 Panel C shows the editing frequency of each mgRNA / hgRNA pair targeting the human LAG3 gene calculated by EditR analysis.

[0127] Figure 14 Indicates the editing efficiency induced by tBE and the mgRNA targeting human CISH and its hgRNA pair. Figure 14 Panel A is a schematic diagram illustrating the co-transfection of the mgRNA targeting the human CISH gene and its different hgRNAs with tBE-V5-mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 14 Panel B shows the editing efficiency induced by tBE-V5-mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 14 Panel C shows the editing frequency of each mgRNA / hgRNA pair targeting the human CISH gene calculated by EditR analysis.

[0128] Figure 15 Is the validation of CISH KO at the protein level. Figure 15 Panels A-C are the flow analysis results of the CISH protein levels in MOCK (A) or cells transfected with tBE + CISH-mg2-U2 (B) or cells transfected with tBE + CISH-mg3-U3 (C) in K562 cells. Figure 15 Panel D is a summary of the CISH+ cell ratios in 15A-C.

[0129] Figure 16 Indicates the editing efficiency induced by tBE and the mgRNA targeting human TGFBR2 and its hgRNA pair. Figure 16 Panel A is a schematic diagram illustrating the co-transfection of the mgRNA targeting the human TGFBR2 gene and its different hgRNAs with tBE-V5-mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 16 Panel B shows the editing efficiency induced by tBE-V5-mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 16 Panel C shows the editing frequency of each mgRNA / hgRNA pair targeting the human TGFBR2 gene calculated by EditR analysis.

[0130] Figure 17 Is the validation of TGFBR2 KO at the protein level. Figure 17Flow cytometry analysis results of TGFBR2 protein levels in MOCK (A), cells transfected with tBE + TGFBR2-mg3-U1 (B), or cells transfected with tBE + TGFBR2-mg4-U1 (C) in NK92 cells. Figure 17 D is a summary of the TGFBR2+ cell ratios in 17A - C.

[0131] Figure 18 Indicates the editing efficiency induced by tBE and the mgRNA and its hgRNA targeting human FAS. Figure 18 A is a schematic diagram illustrating the co - transfection of mgRNA and its different hgRNAs targeting the human FAS gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 18 B shows the editing efficiency induced by tBE - V5 - mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 18 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human FAS gene calculated by EditR analysis.

[0132] Figure 19 Is the verification of FAS KO at the protein level. Figure 19 A - C are flow cytometry analysis results of FAS protein levels in MOCK (A), cells transfected with tBE + FAS - mg1 - U1 (B), or cells transfected with tBE + FAS - mg5 - U1 (C) in Jurkat cells. Figure 19 D is a summary of the FAS+ cell ratios in 19A - C.

[0133] Figure 20 Indicates the editing efficiency induced by tBE and the mgRNA and its hgRNA targeting human CD7. Figure 20 A is a schematic diagram illustrating the co - transfection of mgRNA and its different hgRNAs targeting the human CD7 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 20 B shows the editing efficiency induced by tBE - V5 - mA3 and the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 20 C shows the editing frequency of each mgRNA / hgRNA pair targeting the human CD7 gene calculated by EditR analysis.

[0134] Figure 21 Indicates the editing efficiency induced by tBE and the mgRNA and its hgRNA targeting human CBLB. Figure 21A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human CBLB gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 21 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 21 C shows the editing efficiency of each mgRNA / hgRNA pair targeting the human CBLB gene calculated by EditR analysis.

[0135] Figure 22 is the verification of CBLB KO at the protein level. Figure 22 is the result of Western blot analysis of CBLB protein levels in MOCK (NC) or cells transfected with tBE + CBLB - mg2 - U or cells transfected with tBE + CBLB - mg10 - U1 in NK92 cells.

[0136] Figure 23 represents the editing efficiency induced by tBE with the mgRNA and its hgRNA pair targeting the human KLRC1 gene. Figure 23 A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human KLRC1 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 23 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 23 C shows the editing efficiency of each mgRNA / hgRNA pair targeting the human KLRC1 gene calculated by EditR analysis.

[0137] Figure 24 is the verification of KLRC1 KO at the protein level. Figure 24 A - C are the results of flow cytometry analysis of NKG2A protein levels in MOCK (A) or cells transfected with tBE + KLRC1 - mg2 - U1 (B) or cells transfected with tBE + KLRC1 - mg5 - U1 (C) in NK92 cells. Figure 24 D is a summary of the NKG2A + cell ratio in 24A - C.

[0138] Figure 25 represents the editing efficiency induced by tBE with the mgRNA and its hgRNA pair targeting the human CD38 gene. Figure 25A is a schematic diagram illustrating the co - transfection of mgRNAs and their different hgRNAs targeting the human CD38 gene with tBE - V5 - mA3 (SEQ ID NO:731) and nCas9 (SEQ ID NO:732). Figure 25 B shows the editing efficiency induced by tBE - V5 - mA3 with the indicated mgRNA / hgRNA pairs at the indicated sites. Figure 25 C shows the editing efficiency of the mgRNA / hgRNA pairs targeting the human CD38 gene calculated by EditR analysis.

[0139] Figure 26 is the verification of CD38 KO at the protein level. Figure 26 A - C are the results of flow cytometry analysis of CD38 protein levels in MOCK (A) or cells transfected with tBE + CD38 - mg2 - U2 (B) or cells transfected with tBE + CD38 - mg7 - U1 in NK92 cells. Figure 25 D is a summary of the NKG2A+ cell ratio in 25A - C. Detailed Description of the Invention

[0140] Definition

[0141] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, preferred methods and materials are described herein. Accordingly, the terms defined herein are described more fully by reference to the entire specification.

[0142] As used herein, unless the context clearly indicates otherwise, the singular terms "a", "an", and "the" include plural referents.

[0143] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as combinations lacking when interpreted as an alternative ("or"). In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted.

[0144] Unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" or similar terms mean non - exclusive inclusion, such that a listing of elements or features does not include only those elements or features stated or listed, but may include other elements or features not stated or listed.

[0145] Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction, and amino acid sequences are written left to right in the amino to carboxyl direction. When used in the context of an amino acid sequence, the number "n" refers to the nth amino acid counted from the amino terminus of the amino acid sequence. For example, "amino acid 15" refers to the 15th amino acid in a given amino acid sequence. For example, "R15" refers to the 15th amino acid in a given amino acid sequence being arginine (R).

[0146] It should be understood that the present disclosure is not limited to the specific methods, protocols, and reagents described, as these may vary depending on the context in which those skilled in the art use them.

[0147] As used herein, when applied to nucleic acid or polynucleotide sequences, the terms "percent identity" and "identity %" refer to the percentage of residue matches between at least two nucleic acid or polynucleotide sequences aligned using a standardized algorithm. Such algorithms can insert gaps in the sequences being compared in a standardized and reproducible manner in order to optimize the alignment between the two sequences and thus achieve a more meaningful comparison of the two sequences.

[0148] The percent identity between nucleic acid or polynucleotide sequences can be determined using a general and freely available set of sequence comparison algorithms provided by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) of the United States (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which algorithms are available from several sources, including NCBI, Bethesda, Md., and the Internet at http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0149] However, due to the degeneracy of the genetic code, nucleic acid or polynucleotide sequences that do not display a high degree of identity can encode similar amino acid sequences. It should be understood that this degeneracy can be exploited to alter nucleic acid sequences to produce multiple nucleic acid sequences that all encode substantially the same protein. Specifically, degenerate codon substitutions can be achieved by generating sequences in which one or more of the selected (or all) codons have their third positions substituted with a mixed base and / or deoxyinosine residue (Batzer et al. (1991) Nucleic Acid Res 19:5081; Ohtsuka et al. 1985) J Biol Chem 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98). The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term nucleic acid can be used interchangeably with polynucleotide and (in the appropriate context) gene, cDNA, and mRNA encoded by a gene.

[0150] As used herein, "percent amino acid sequence identity (%)" with respect to a peptide, polypeptide, or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in another peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The percent amino acid sequence identity in the present disclosure is measured using BLAST software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0151] An amino acid substitution refers to the replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions can be conservative or non-conservative substitutions. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the protein of interest, and the products can be screened for the desired activity (e.g., retained / improved biological activity).

[0152] Table 1

[0153]

[0154]

[0155] Amino acids can be grouped according to common side-chain properties:

[0156] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0157] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0158] (3) Acidic: Asp, Glu;

[0159] (4) Basic: His, Lys, Arg;

[0160] (5) Residues affecting chain direction: Gly, Pro;

[0161] (6) Aromatic: Trp, Tyr, Phe.

[0162] As used herein, the term "polypeptide" is intended to cover both a single "polypeptide" and plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to a specific length of the product. Thus, "peptide", "protein", or any other term used to refer to one or more chains of two or more amino acids is included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or interchangeably with any one of these terms. The term "polypeptide" also means the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a designated nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

[0163] As used herein, when applied to a polynucleotide, the term "encode" or "encoding" means that a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, the polynucleotide can be transcribed and / or translated to produce an mRNA of the polypeptide and / or its fragment. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0164] "Guide RNA" (gRNA) refers to a synthetic or expressed RNA sequence that contains a CRISPR binding motif and a spacer. In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is a dual RNA structure. In some embodiments, the guide RNA is a dual RNA structure formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a LigoRNA. A "spacer" is a DNA targeting motif, a sequence that is complementary to a specific DNA region of the target. In some embodiments, the guide RNA is a crRNA-tracrRNA dual RNA structure, and the crRNA contains the spacer. The CRISPR binding motif of the guide RNA can bind to a Cas enzyme, and the DNA targeting motif of the gRNA can direct the complex to a specific target location on the DNA. In some embodiments, the guide RNA is a crRNA-tracrRNA dual RNA structure, and the base pair structure formed by the crRNA and the tracrRNA contains the CRISPR binding motif. The guide RNA can further contain one or more protein binding motifs.

[0165] As used herein, a "fusion protein" is a protein that contains at least two domains encoded by separate genes that have been linked into a single polypeptide. For example, a fusion protein can contain two domains encoded by separate genes that have been linked such that they are transcribed and translated as a single unit, yielding a single polypeptide. In some embodiments, the at least two domains are directly fused together. In some embodiments, the domains are linked by one or more linkers.

[0166] As used herein, the term "genetic modification" and its grammatical equivalents can refer to one or more alterations of nucleic acids, such as nucleic acids in the genome of an organism. For example, genetic modification can refer to an alteration, addition, and / or deletion of a gene or a portion of a gene or other nucleic acid sequence. A genetically modified cell can also refer to a cell that has an added, deleted, and / or altered gene or portion of a gene. A genetically modified cell can also refer to a cell that has an added nucleic acid sequence that is not a gene or a portion of a gene. Genetic modification includes, for example, transient knock-in or knockdown mechanisms, as well as mechanisms that result in permanent knock-in, knockdown, or knockout of a target gene or portion of a gene or nucleic acid sequence. Genetic modification includes, for example, transient knock-in and mechanisms that result in permanent knock-in of a nucleic acid sequence. Genetic modification also includes, for example, decreased or increased transcription, decreased or increased mRNA stability, decreased or increased translation, and decreased or increased protein stability.

[0167] As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells).

[0168] The term "subject" means any animal, such as a mammal, e.g., a human.

[0169] As used herein, the terms "treat", "treating", or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder, or reducing at least one of its clinical symptoms. For example, in some embodiments, ameliorating a disease or disorder can include obtaining a beneficial or desired clinical outcome, which includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the severity of the disease, preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the state of the disease, inhibiting or eliminating the disease or the progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).

[0170] As used herein, for a given subject, "allogeneic" cells refer to cells obtained from a different individual of the same species as the subject and are genetically different from cells obtained from the given subject.

[0171] As used herein, the term "immunogenicity" refers to the ability or tendency of a substance to elicit an unwanted immune response against itself in a subject.

[0172] Immunogenicity of allogeneic cells

[0173] Allogeneic cell therapy typically faces two major challenges. First, the administered allogeneic cells can cause life-threatening graft-versus-host disease (GvHD). Second, these allogeneic cells can be rapidly eliminated by the host immune system, limiting the persistence of their biological activity.

[0174] Graft-versus-host disease (GvHD) is a systemic disease that occurs when the immune cells of the graft recognize the host as foreign and attack the recipient's somatic cells. "Graft" refers to the transplanted or donated tissue, and "host" refers to the recipient's tissue. GvHD is one of the main causes of death after allogeneic hematopoietic stem cell transplantation and must therefore be prevented. Since T cell alloreactivity depends on the interaction of the T cell receptor (TCR) with the alloantigen presented by human leukocyte antigen (HLA), TCR-depleted T cells do not cause GvH reactions when infused into HLA-mismatched patients. Many research groups are working on reducing the risk of GvHD by genetically ablating the TCR locus (primarily the TCRα constant region (TRAC, human TRAC: ENMG00000277734)), which can effectively reduce the risk of GvHD and the GvH reactions involved in giving allogeneic cells to a subject.

[0175] On the other hand, to prevent or reduce host immune rejection, one approach is to reduce the immunogenicity of allogeneic T cells, for example, by genetically abolishing the beta-2 microglobulin (B2M) gene (human B2M: ENMG00000166710) to disrupt MHC class I molecules. Another way is to delete CD52 of donor T cells (human CD52: ENMG00000169442) and use anti-CD52 monoclonal antibodies to eliminate host T cells (which express CD52) to avoid allogeneic rejection. In addition, inhibitory checkpoints (such as, PD-1, encoded by the PDCD1 gene, human PDCD1: ENMG00000188389) can be knocked out separately or simultaneously with TRAC, CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and / or CD38 to enhance the potency and persistence of autologous or allogeneic CAR-T cells or NK cells (Depil, S., et al. "‘Off-the-shelf’allogeneic CAR T cells: development and challenges." Nature reviews Drug discovery 19.3 (2020): 185-199; Jung, In-Young, and Jungmin Lee. "Unleashing the therapeutic potential of CAR-T cell therapy using gene-editing technologies." Molecules and Cells 41.8 (2018): 717; Lin, Haolong, et al. "Advances in universal CAR-T cell therapy." Frontiers in Immunology (2021): 4014). For anti-CD7 or anti-CD38 CAR-T cells, CD7 or CD38 can be knocked out to prevent fratricide. The FAS / FASL signal induces apoptosis of cytotoxic T cells, which weakens the anti-tumor efficacy of CAR-T therapy. TGF-β secreted in the tumor microenvironment (TME) inhibits T cell function by binding to TGFBR2. Cytokine-induced SH2 (CISH) protein is induced in CD8+ T cells after TCR stimulation and inhibits T cell anti-tumor function. CISH is also a key negative feedback regulator of IL-15 signaling in NK cells. CBLB has been characterized as an intracellular checkpoint in T cells as well as NK cells, and deletion of CBLB enhances the function of T cells and NK cells.The KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is an effective NK cell immune checkpoint.

[0176] The present disclosure provides that reducing or preventing host immune rejection in a subject, or promoting the survival and persistence of therapeutic cells, can be achieved by disrupting (e.g., by knockdown or knockout) the expression of the following genes alone or in combination. The genes are TCRα constant gene (TRAC, human TRAC: ENMG00000277734), b2-microglobulin (B2M) gene (human B2M: ENMG00000166710), CD52 gene (human CD52: ENMG00000169442), PDCD1 gene (human PDCD1: ENMG00000188389), cytotoxic T lymphocyte-associated protein 4 gene (CTLA4, ENMG00000163599), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain-containing gene (TIGIT, ENMG00000181847), hepatitis A virus cellular receptor 2 gene (HAVCR2 / TIM3, ENMG00000135077), lymphocyte activation gene 3 (LAG3, ENMG00000089692), cytokine-inducible SH2-containing protein gene (CISH, ENMG00000114737), transforming growth factor β receptor 2 gene (TGFBR2, ENMG00000163513), Fas cell surface death receptor gene (FAS, ENMG00000026103), CD7 gene (ENMG00000173762), Cbl proto-oncogene B (CBLB) gene (human CBLB: ENSG00000114423), killer cell lectin-like receptor C1 (KLRC1) gene (human KLRC1: ENSG00000134545), and CD38 gene (human CD38: ENSG00000004468).

[0177] Gene editing system

[0178] The safety and efficiency of gene editing tools are very important in clinical applications. Previous studies have reported that Cas9 nuclease-induced DSBs can activate the p53-mediated DDR pathway and subsequently lead to cell death. In addition, APOBEC / AID family members can trigger C-to-T base substitutions in single-stranded DNA (ssDNA) regions, which randomly form during various cellular processes including DNA replication, repair, and transcription. Therefore, the specificity of previous base editing systems is compromised, limiting the application of base editors (BEs) for therapeutic purposes.

[0179] In the present disclosure, a newly developed variant of the base editing system, the transformer base editor (tBE), is used. The tBE can specifically edit cytosine in the target region without observable off-target mutations.

[0180] In some embodiments, the transformer base editor (tBE) system comprises a deoxycytidine deaminase inhibitor (dCDI) domain and a split TEV protease. Thus, the tBE remains inactive at off-target sites with cleavable fused dCDI domains and eliminates unintended off-target mutations. Only when bound at the on-target site is the tBE converted to cleave the dCDI domain and catalyze targeted deamination for precise editing. Specifically, the tBE uses a single guide RNA (sgRNA, typically 20 nt) to bind at the target genomic locus and an auxiliary sgRNA (asgRNA, typically 10 to 20 nt) to bind in a nearby region (preferably upstream of the target genomic locus). The binding of these two gRNAs can direct the components of the tBE system to correctly assemble at the target genomic locus for base editing. The tBE can specifically edit cytosine in the target region without observable off-target mutations, for example, inducing premature stop codons to inhibit target protein expression or disrupting the GU-AG consensus sequence to disrupt splicing sites. In addition, when using Cas9 nickase (D10A), the tBE system is less toxic to cells than Cas9 nuclease because Cas9 nickase activates a lower level of p53-mediated DDR.

[0181] The present disclosure provides that the tBE system can be used to disrupt the TRAC, B2M, CD52, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, or CD38 genes in cells, either alone or in combination, to prevent GvHD or reduce the immunogenicity of the cells, thereby enhancing the expansion and persistence of CAR-T cells or other therapeutic cells after infusion. In some embodiments, the cells are human immune cells, such as human T cells and natural killer (NK) cells.

[0182] In some embodiments, the tBE is used for the genetic engineering of human T cells, NK cells, and other immune cells to construct allogeneic or enhanced autologous chimeric antigen receptor T (CAR-T) cells and other cell therapy products in clinical applications. In some embodiments, the tBE is used to induce efficient and precise gene editing at genomic loci to disrupt genes associated with graft-versus-host disease (GvHD), allogeneic rejection by the host, immunosuppression, or T cell fratricide.

[0183] In some embodiments, a highly specific base editing system, a variant base editor (tBE), is used, which can edit cytosine in the target region without observable off-target mutations. In some embodiments, the tBE is any of the base editors described in WO2020156575A1, which is incorporated herein by reference in its entirety. For example, the tBE can be any base editor as shown in Figure 1 any of those shown in

[0184] The present disclosure provides various combinations of guide RNAs (mgRNAs) and helper mgRNAs (hgRNAs) with high editing efficiency for target genes: the T cell receptor alpha constant (TRAC) gene, the beta-2 microglobulin (B2M) gene, the CD52 gene, the programmed cell death 1 (PDCD1) gene, the cytotoxic T lymphocyte-associated protein 4 (CTLA4) gene, the T cell immunoreceptor with Ig and ITIM domains (TIGIT) gene, the hepatitis A virus cellular receptor 2 (HAVCR2 / TIM3) gene, the lymphocyte activation 3 (LAG3) gene, the cytokine-inducible SH2-containing protein (CISH) gene, the transforming growth factor beta receptor 2 (TGFBR2) gene, the Fas cell surface death receptor (FAS) gene, the CD7 gene, the Cbl proto-oncogene B (CBLB) gene, the killer cell lectin-like receptor C1 (KLRC1) gene, and the CD38 gene. In some embodiments, these mgRNA / hgRNA pairs can be used to construct allogeneic or enhanced autologous CAR-T cells, or other types of allogeneic or enhanced cell therapies in clinical applications.

[0185] The base editors, the combinations of mgRNA / hgRNA, and the base editing methods provided herein can be applied to perform highly specific and efficient base editing in the genomes of various eukaryotes. They achieve high specificity and efficiency at most sites. The present disclosure enhances the clinical translation of tBE, particularly for constructing allogeneic or enhanced autologous CAR-T cells and other types of allogeneic or enhanced cell therapies.

[0186] In some embodiments, the base editor as used herein is a cytosine base editor (CBE), which comprises a combination of a CRISPR system and a cytidine deaminase. The CBE achieves programmable cytosine-to-thymine (C-to-T) substitution. Since the base editing process does not rely on the generation of DNA double-strand breaks (DSBs), unwanted nucleotide insertions / deletions (indels) or DNA damage responses (DDRs) can be largely avoided.

[0187] In some embodiments, the gene editing systems disclosed herein disrupt a target gene by generating a stop codon or disrupting a splice site in the target gene.

[0188] In some embodiments, the gene editing systems disclosed herein induce a C-to-T base edit in the codons CAA (Gln), CAG (Gln), TGG (Trp, C to T on the opposite strand), or CGA (Arg) in the target gene to generate the TAA, TAG, or TGA stop codons.

[0189] In some embodiments, the gene editing systems disclosed herein induce a G-to-A (C to T on the opposite strand) base edit in the GT or AG splice sites to disrupt the GU-AG canonical splicing pattern.

[0190] In some embodiments, the present disclosure provides a gene editing system for disrupting the TRAC gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the TRAC gene. In some embodiments, a highly specific base editor (variant base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the TRAC gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the TRAC gene.

[0191] In some embodiments, the present disclosure provides a gene editing system for disrupting the B2M gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the B2M gene. In some embodiments, a highly specific base editor (variant base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the B2M gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the B2M gene.

[0192] In some embodiments, the present disclosure provides a gene editing system for disrupting the CD52 gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CD52 gene. In some embodiments, a highly specific base editor (variant base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CD52 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CD52 gene.

[0193] In some embodiments, the present disclosure provides a gene editing system for disrupting the PDCD1 gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the PDCD1 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the PDCD1 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the PDCD1 gene.

[0194] In some embodiments, the present disclosure provides a gene editing system for disrupting the CTLA4 gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CTLA4 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CTLA4 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CTLA4 gene.

[0195] In some embodiments, the present disclosure provides a gene editing system for disrupting the TIGIT gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the TIGIT gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the TIGIT gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the TIGIT gene.

[0196] In some embodiments, the present disclosure provides a gene editing system for disrupting the TIM3 gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the TIM3 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the TIM3 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the TIM3 gene.

[0197] In some embodiments, the present disclosure provides a gene editing system for disrupting LAG3, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the LAG3 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the LAG3 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the LAG3 gene.

[0198] In some embodiments, the present disclosure provides a gene editing system for disrupting the CISH gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CISH gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CISH gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CISH gene.

[0199] In some embodiments, the present disclosure provides a gene editing system for disrupting TGFBR2, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the TGFBR2 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the TGFBR2 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the TGFBR2 gene.

[0200] In some embodiments, the present disclosure provides a gene editing system for disrupting the FAS gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the FAS gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the FAS gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the FAS gene.

[0201] In some embodiments, the present disclosure provides a gene editing system for disrupting CD7, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CD7 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CD7 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CD7 gene.

[0202] In some embodiments, the present disclosure provides a gene editing system for disrupting the CBLB gene, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CBLB gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CBLB gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CBLB gene.

[0203] In some embodiments, the present disclosure provides a gene editing system for disrupting KLRC1, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the KLRC1 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the KLRC1 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the KLRC1 gene.

[0204] In some embodiments, the present disclosure provides a gene editing system for disrupting CD38, wherein the gene editing system comprises a base editor and at least one guide RNA capable of binding to the CD38 gene. In some embodiments, a highly specific base editor (transformed base editor (tBE)) is used to induce efficient and precise gene editing at a genomic locus to disrupt the CD38 gene. The tBE comprises a combination of a guide RNA (mgRNA) and an auxiliary mgRNA (hgRNA), wherein the mgRNA and hgRNA are capable of binding to the CD38 gene.

[0205] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the T cell receptor alpha constant (TRAC) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 1-5.

[0206] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 2.

[0207] Table 2. Combinations of mgRNA spacer and hgRNA spacer (TRAC)

[0208] mgRNA hgRNA SEQ ID NO:01 SEQ ID NO:39 SEQ ID NO:01 SEQ ID NO:40 SEQ ID NO:01 SEQ ID NO:41 SEQ ID NO:02 SEQ ID NO:39 SEQ ID NO:02 SEQ ID NO:40 SEQ ID NO:02 SEQ ID NO:41 SEQ ID NO:03 SEQ ID NO:42 SEQ ID NO:03 SEQ ID NO:43 SEQ ID NO:03 SEQ ID NO:44 SEQ ID NO:04 SEQ ID NO:45 SEQ ID NO:04 SEQ ID NO:46 SEQ ID NO:04 SEQ ID NO:47 SEQ ID NO:05 SEQ ID NO:48 SEQ ID NO:05 SEQ ID NO:49 SEQ ID NO:05 SEQ ID NO:50

[0209] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0210] Table 26

[0211] mgRNA hgRNA SEQ ID NO:679 SEQ ID NO:680 SEQ ID NO:679 SEQ ID NO:681 SEQ ID NO:682 SEQ ID NO:684 SEQ ID NO:682 SEQ ID NO:685; or SEQ ID NO:683 SEQ ID NO:685

[0212] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD52 gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 6-8.

[0213] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 3.

[0214] Table 3. Combinations of mgRNA spacer and hgRNA spacer (CD52)

[0215]

[0216]

[0217] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0218] Table 27

[0219] mgRNA hgRNA SEQ ID NO:686 SEQ ID NO:688 SEQ ID NO:687 SEQ ID NO:688; or SEQ ID NO:689 SEQ ID NO:690

[0220] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the beta-2 microglobulin (B2M) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 9-19.

[0221] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 4.

[0222] Table 4. Combinations of mgRNA spacer and hgRNA spacer (B2M)

[0223]

[0224]

[0225]

[0226] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0227] Table 28

[0228] mgRNA hgRNA SEQ ID NO:697 SEQ ID NO:698; or SEQ ID NO:699 SEQ ID NO:700

[0229] In another aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the programmed cell death protein 1 (PDCD1) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 20-38.

[0230] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 5.

[0231] Table 5. Combinations of mgRNA spacer and hgRNA spacer (PDCD1)

[0232]

[0233]

[0234] In some embodiments, the nucleic acid sequences of the mgRNA and hgRNA respectively comprise:

[0235] Table 29

[0236] mgRNA hgRNA SEQ ID NO:691 SEQ ID NO:692 SEQ ID NO:693 SEQ ID NO:694; or SEQ ID NO:695 SEQ ID NO:696

[0237] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytotoxic T lymphocyte-associated protein 4 (CTLA4) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 247-256.

[0238] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 13.

[0239] Table 13

[0240] mgRNA hgRNA SEQ ID NO:247 SEQ ID NO:257 SEQ ID NO:247 SEQ ID NO:258 SEQ ID NO:248 SEQ ID NO:259 SEQ ID NO:249 SEQ ID NO:260 SEQ ID NO:249 SEQ ID NO:261 SEQ ID NO:250 SEQ ID NO:262 SEQ ID NO:250 SEQ ID NO:263 SEQ ID NO:250 SEQ ID NO:264 SEQ ID NO:251 SEQ ID NO:265 SEQ ID NO:251 SEQ ID NO:266 SEQ ID NO:251 SEQ ID NO:267 SEQ ID NO:252 SEQ ID NO:268 SEQ ID NO:252 SEQ ID NO:269 SEQ ID NO:253 SEQ ID NO:268 SEQ ID NO:253 SEQ ID NO:269 SEQ ID NO:253 SEQ ID NO:270 SEQ ID NO:254 SEQ ID NO:271 SEQ ID NO:254 SEQ ID NO:272 SEQ ID NO:255 SEQ ID NO:273 SEQ ID NO:255 SEQ ID NO:274 SEQ ID NO:255 SEQ ID NO:275 SEQ ID NO:256 SEQ ID NO:276 SEQ ID NO:256 SEQ ID NO:277

[0241] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the T cell immunoreceptor with Ig and ITIM domains (TIGIT) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 278-294.

[0242] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 14.

[0243] Table 14

[0244]

[0245]

[0246] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the hepatitis A virus cellular receptor 2 (HAVCR2 / TIM3) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 323 - 337.

[0247] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 15.

[0248] Table 15

[0249]

[0250]

[0251]

[0252] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the lymphocyte activation 3 (LAG3) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 364 - 396.

[0253] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 16.

[0254] Table 16

[0255]

[0256]

[0257]

[0258] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytokine-inducible SH2-containing protein (CISH) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 472-482.

[0259] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 17.

[0260] Table 17

[0261]

[0262]

[0263] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0264] Table 30

[0265] mgRNA hgRNA SEQ ID NO:701 SEQ ID NO:702 SEQ ID NO:701 SEQ ID NO:703 SEQ ID NO:704 SEQ ID NO:705 SEQ ID NO:704 SEQ ID NO:706; or SEQ ID NO:707 SEQ ID NO:708

[0266] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or said hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the transforming growth factor beta receptor 2 (TGFBR2) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 504-510.

[0267] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 18.

[0268] Table 18

[0269]

[0270]

[0271] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0272] Table 31

[0273] mgRNA hgRNA SEQ ID NO:709 SEQ ID NO:710 SEQ ID NO:709 SEQ ID NO:711 SEQ ID NO:712 SEQ ID NO:713; or SEQ ID NO:712 SEQ ID NO:714

[0274] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the Fas cell surface death receptor (FAS) gene and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 530 - 541.

[0275] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 19.

[0276] Table 19

[0277]

[0278]

[0279] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0280] Table 32

[0281] mgRNA hgRNA SEQ ID NO:715 SEQ ID NO:716; or SEQ ID NO:717 SEQ ID NO:718

[0282] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or the hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD7 gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 565 - 575.

[0283] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 20.

[0284] Table 20

[0285]

[0286]

[0287] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CBLB gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 609 - 618.

[0288] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 21.

[0289] Table 21

[0290]

[0291] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0292] Table 33

[0293] mgRNA hgRNA SEQ ID NO:723 SEQ ID NO:724; or SEQ ID NO:725 SEQ ID NO:726

[0294] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the KLRC1 gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 637 - 641.

[0295] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 22.

[0296] Table 22

[0297] mgRNA hgRNA SEQ ID NO.637 SEQ ID NO.642 SEQ ID NO.637 SEQ ID NO.643 SEQ ID NO.638 SEQ ID NO.644 SEQ ID NO.638 SEQ ID NO.645 SEQ ID NO.639 SEQ ID NO.646 SEQ ID NO.639 SEQ ID NO.647 SEQ ID NO.640 SEQ ID NO.648 SEQ ID NO.640 SEQ ID NO.649 SEQ ID NO.641 SEQ ID NO.650

[0298] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0299] Table 34

[0300] mgRNA hgRNA SEQ ID NO:727 SEQ ID NO:728; or SEQ ID NO:729 SEQ ID NO:730

[0301] In one aspect, the present disclosure provides a gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the CD38 gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 651-659.

[0302] In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences shown in Table 23.

[0303] Table 23

[0304] mgRNA hgRNA SEQ ID NO.651 SEQ ID NO.660 SEQ ID NO.651 SEQ ID NO.661 SEQ ID NO.651 SEQ ID NO.662 SEQ ID NO.652 SEQ ID NO.663 SEQ ID NO.652 SEQ ID NO.664 SEQ ID NO.652 SEQ ID NO.665 SEQ ID NO.653 SEQ ID NO.666 SEQ ID NO.654 SEQ ID NO.667 SEQ ID NO.655 SEQ ID NO.668 SEQ ID NO.655 SEQ ID NO.669 SEQ ID NO.655 SEQ ID NO.670 SEQ ID NO.656 SEQ ID NO.671 SEQ ID NO.657 SEQ ID NO.672 SEQ ID NO.657 SEQ ID NO.673 SEQ ID NO.658 SEQ ID NO.674 SEQ ID NO.658 SEQ ID NO.675 SEQ ID NO.659 SEQ ID NO.676 SEQ ID NO.659 SEQ ID NO.677 SEQ ID NO.659 SEQ ID NO.678

[0305] In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

[0306] Table 35

[0307] mgRNA hgRNA SEQ ID NO:719 SEQ ID NO:720; or SEQ ID NO:721 SEQ ID NO:722

[0308] In some embodiments, the gene editing system described herein comprises: a first mgRNA comprising a first mgRNA spacer targeting a first gene and a second mgRNA comprising a second mgRNA spacer targeting a second gene, wherein the first gene and the second gene are each independently selected from: the TRAC gene, the B2M gene, the CD52 gene, the PDCD1 gene, the CTLA4 gene, the TIGIT gene, the TIM3 gene, the LAG3 gene, the CISH gene, the TGFBR2 gene, the FAS gene, the CD7 gene, the CBLB gene, the KLRC1 gene, and the CD38 gene. In some embodiments, the first gene and the second gene are different.

[0309] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, and (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, and wherein the first Cas protein and the second Cas protein are the same or different.

[0310] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, and (7) a nucleobase deaminase inhibitor domain, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain.

[0311] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising the protease and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the protease and the second RNA-binding domain are optionally linked by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA-binding domain binds to the second protein-binding motif.

[0312] In some embodiments, the protease is split into a first protease fragment and a second protease fragment, wherein neither the first nor the second protease fragment alone can cleave the cleavage site.

[0313] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment is optionally linked to the second RNA-binding domain by a linker, and (9) a third fusion protein comprising a second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA-binding domain binds to the second protein-binding motif, and wherein the third RNA-binding domain binds to the third protein-binding motif.

[0314] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment is optionally linked to the second RNA-binding domain by a linker, and (9) a third fusion protein comprising a second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA-binding domain binds to the second protein-binding motif, wherein the third RNA-binding domain binds to the third protein-binding motif, and wherein the second and third RNA-binding domains are the same or different, and the second and third protein-binding motifs are the same or different.

[0315] In some embodiments, the gene editing systems disclosed herein comprise: (1) an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain is optionally linked to the first RNA-binding domain by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and the second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA-binding domain binds to the second protein-binding motif.

[0316] "Protease" refers to an enzyme that catalyzes proteolysis. "Cleavage site for protease" refers to a short peptide recognized by a protease and that generates a proteolytic cleavage within the short peptide. Non-limiting examples of proteases include TEV protease, TuMV protease, PPV protease, PVY protease, ZIKV protease, and WNV protease. The protein sequences of exemplary proteases and their corresponding cleavage sites are provided in Table 6.

[0317] Table 6. Exemplary Proteases and Their Cleavage Sites

[0318]

[0319]

[0320]

[0321] In some embodiments, the protease is TEV protease, TuMV protease, PPV protease, PVY protease, ZIKV protease or WNV protease.

[0322] In some embodiments, the protease cleavage site is a self-cleaving peptide, such as a 2A peptide. A "2A peptide" is a viral oligopeptide that is 18-22 amino acids long and mediates "cleavage" of polypeptides during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viral 2As are typically named after the virus from which they are derived. The first 2A discovered was F2A (foot-and-mouth disease virus), followed by E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A). Some non-limiting examples of 2A peptides are provided in SEQ ID NO: 138-140.

[0323] In some embodiments, the protease is TEV protease. In some embodiments, the TEV protease comprises the sequence shown in SEQ ID NO: 124.

[0324] In some embodiments, the first and / or second TEV protease fragment alone cannot cleave the TEV cleavage site. However, in the presence of the remaining portion of the TEV protease, this fragment is capable of effecting cleavage. The TEV fragment can be the TEV N-terminal domain (such as SEQ ID NO: 125) or the TEV C-terminal domain (such as SEQ ID NO: 126). In some embodiments, the first TEV protease fragment comprises the sequence of SEQ ID NO: 125. In some embodiments, the first TEV protease fragment comprises the sequence of SEQ ID NO: 126.

[0325] A "nucleobase deaminase inhibitor" or "inhibitory domain" refers to a protein or protein domain that inhibits the deaminase activity of a nucleobase deaminase.

[0326] In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of a nucleobase deaminase.

[0327] In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of a cytidine deaminase. In some embodiments, the nucleobase deaminase inhibitor is the mouse APOBEC3 cytidine deaminase domain 2 (mA3-CDA2, SEQ ID NO: 141). In some embodiments, the nucleobase deaminase inhibitor is the human APOBEC3B cytidine deaminase domain 1 (hA3B-CDA1, SEQ ID NO: 142).

[0328] Table 7 shows 44 proteins / domains with significant sequence homology to the mA3-CDA2 core sequence, and Table 8 shows 43 proteins / domains with significant sequence homology to hA3B-CDA1. All of these proteins and domains, as well as their variants and equivalents, are considered to have nucleobase deaminase inhibitory activity.

[0329] Table 7

[0330]

[0331]

[0332]

[0333] Table 8

[0334]

[0335]

[0336]

[0337]

[0338] In some embodiments, the inhibitory domain of the cytidine deaminase comprises the amino acid sequence shown in SEQ ID NO:141 or SEQ ID NO:142.

[0339] As used herein, the term "nucleobase deaminase" refers to a group of enzymes that catalyze the hydrolytic deamination of nucleobases such as cytidine, deoxycytidine, adenosine, and deoxyadenosine. Non-limiting examples of nucleobase deaminases include cytidine deaminase and adenosine deaminase.

[0340] Some nucleobase deaminases have a single catalytic domain, while others also have other domains, such as the inhibitory domain described in WO2020156575A1. Thus, in some embodiments, the gene editing systems disclosed herein include only the catalytic domain, such as the murine A3 cytidine deaminase domain 1 (mA3-CDA1, SEQ ID NO:143) and the human A3B cytidine deaminase domain 2 (hA3B-CDA2, SEQ ID NO:144). In some embodiments, the gene editing systems disclosed herein include at least the catalytic core of the catalytic domain. For example, when mA3-CDA1 is truncated at residues 196 / 197, the CDA1 domain still retains substantial editing efficiency.

[0341] In some embodiments, the nucleotide deaminase is a cytidine deaminase. In some embodiments, the nucleotide deaminase is a cytidine deaminase comprising the amino acid sequence of SEQ ID NO:143. In some embodiments, the nucleotide deaminase is a cytidine deaminase comprising the amino acid sequence of SEQ ID NO:144.

[0342] Table 9

[0343]

[0344] "Cytidine deaminase" refers to the enzyme that catalyzes the hydrolysis and deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Cytidine deaminase maintains the cellular pyrimidine pool. The family of cytidine deaminases is APOBEC ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like"). Members of this family are C-to-U editing enzymes. Some APOBEC family members have two domains, one domain of the APOBEC-like protein is the catalytic domain, and the other domain is the pseudo-catalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. The RNA editing of APOBEC-1 requires homodimerization, and this complex interacts with an RNA-binding protein to form an editosome.

[0345] Non-limiting examples of APOBEC proteins include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase (AID).

[0346] Various mutants of APOBEC proteins are also known to confer different editing characteristics to base editors. For example, for human APOBEC3A, certain mutants (such as W98Y, Y130F, Y132D, W104A, D131Y, and P134Y) are even superior to wild-type human APOBEC3A in terms of editing efficiency or editing window. Thus, the term APOBEC and each of its family members also encompasses variants and mutants that have a certain level (such as 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) of sequence identity with the corresponding wild-type APOBEC protein or catalytic domain and retain cytidine deaminase activity. The variants and mutants can be derived from amino acid additions, deletions, and / or substitutions. In some embodiments, such substitutions are conservative substitutions.

[0347] In some embodiments, the cytidine deaminase is selected from APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), APOBECCI (A1), APOBEC3 (A3), APOBEC2 (A2), APOBEC4 (A4), and AICDA (AID).

[0348] In some embodiments, the cytidine deaminase is a human or murine cytidine deaminase.

[0349] In some embodiments, the catalytic domain of the cytidine deaminase is the murine A3 cytidine deaminase domain 1 (CDA1) or the human A3B cytidine deaminase domain 2 (CDA2).

[0350] In some embodiments, the cytidine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 792 - 827. (Table 24)

[0351] Table 24

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] In some embodiments, the nucleotide deaminase is adenosine deaminase. In some embodiments, the adenosine deaminase comprises the sequences of SEQ ID NOs: 828 - 920.

[0359] In some embodiments, the first fusion protein further comprises a uracil glycosylase inhibitor (UGI).

[0360] "Uracil Glycosylase Inhibitor" (UGI) is a small protein (9.5 kDa) that can be prepared from Bacillus subtilis phage PBS1 and inhibits Escherichia coli (E. coli) uracil-DNA glycosylase (UDG) and UDG from other species. Inhibition of UDG occurs through reversible protein binding with a UDG:UGI stoichiometry of 1:1. UGI is capable of dissociating the UDG-DNA complex. Non-limiting examples of UGI can be found in Bacillus phage AR9 (YP_009283008.1). In some embodiments, UGI comprises the amino acid sequence of SEQ ID NO:145 or has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:145 and retains uracil glycosylase inhibitory activity.

[0361] In some embodiments, the first fusion protein further comprises a nuclear localization sequence (NLS).

[0362] "Nuclear localization signal or sequence" (NLS) is an amino acid sequence that tags a protein for import into the cell nucleus via nuclear transport. Generally, the signal consists of one or more short sequences of positively charged lysine or arginine residues that are exposed on the surface of the protein. Different nuclear localization proteins may share the same NLS. A non-limiting example of an NLS is the internal SV40 nuclear localization sequence (iNLS).

[0363] In some embodiments, a peptide linker is optionally provided between each fragment in any fusion protein. In some embodiments, the peptide linker has 1 to 100 amino acid residues (or 3 - 20, 4 - 15, non-limiting). In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acid residues of the peptide linker are amino acid residues selected from alanine, glycine, cysteine, and serine.

[0364] The term "Cas protein" or "clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein" refers to an RNA-guided DNA endonuclease associated with the CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system in Streptococcus pyogenes and other bacteria. Cas proteins include Cas9 protein, Cas12a (Cpf1) protein, Cas12b (formerly called C2c1) protein, Cas13 protein, and various engineered counterparts. Exemplary Cas proteins include SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQRSpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHAFnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, LsCasl2b, RfCas13d, LwaCas13a, PspCas13b, guCasl3b, and RanCasl3b and those provided in Table 10 below.

[0365] Table 10. Exemplary Cas Proteins

[0366]

[0367]

[0368] In some embodiments, the Cas protein comprises an amino acid sequence of any one of SEQ ID NOs: 733 - 784. (Table 25)

[0369] Table 25

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403] In some embodiments, the Cas protein is Cas9, dead Cas9 (dCas9), or Cas9 nickase (nCas9).

[0404] In some embodiments, the Cas protein is nCas9. In some embodiments, the nCas9 protein is the nCas9-D10A protein. In some embodiments, the nCas9-D10A protein has the sequence of SEQ ID NO:146.

[0405] In some embodiments, the first protein-binding RNA motif and the first RNA-binding domain, the second protein-binding RNA motif and the second RNA-binding domain, and the third protein-binding RNA motif and the third RNA-binding domain are each independently selected from the MS2 phage operator stem-loop and the MS2 coat protein (MCP) or its RNA-binding segment; BoxB and N22P or its RNA-binding segment; the telomerase Ku-binding motif and the Ku protein or its RNA-binding segment; the telomerase Sm7-binding motif and the Sm7 protein or its RNA-binding segment; the PP7 phage operator stem-loop and the PP7 coat protein (PCP) or its RNA-binding segment; the SfMu phage Com stem-loop and the Com RNA-binding protein or its RNA-binding segment; and non-natural RNA aptamers and the corresponding aptamer ligands or their RNA-binding segments. See Table 11.

[0406] Table 11

[0407]

[0408]

[0409]

[0410] For any protein of the present disclosure, its bioequivalents are also provided. In some embodiments, the bioequivalent has at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference protein. Preferably, the bioequivalent retains the desired activity of the reference protein. In some embodiments, the bioequivalent is derived by including one, two, three, four, five or more amino acid additions, deletions, substitutions or combinations thereof. In some embodiments, the substitution is a conservative amino acid substitution.

[0411] In another aspect, the present disclosure provides a kit comprising one or more gene editing systems targeting the PDCD1 gene, the TRAC gene, the B2M gene, the CD52 gene, the CTLA4 gene, the TIGIT gene, the TIM3 gene, the LAG3 gene, the CISH gene, the TGFBR2 gene, the FAS gene, the CD7 gene, the CBLB gene, the KLRC1 gene and / or the CD38 gene.

[0412] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting the TRAC gene, the B2M gene, the CD52 gene, the CTLA4 gene, the TIGIT gene, the TIM3 gene, the LAG3 gene, the CISH gene, the TGFBR2 gene, the FAS gene, the CD7 gene, the CBLB gene, the KLRC1 gene and / or the CD38 gene.

[0413] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TRAC gene and a second gene editing system targeting a gene selected from CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB and CD38.

[0414] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CD52 gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1 and CD38.

[0415] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the B2M gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1 and CD38.

[0416] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting a gene selected from CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0417] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CTLA4 gene and a second gene editing system targeting a gene selected from TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0418] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TIGIT gene and a second gene editing system targeting a gene selected from TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 genes.

[0419] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TIM3 gene and a second gene editing system targeting a gene selected from LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0420] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the LAG3 gene and a second gene editing system targeting a gene selected from CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0421] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CISH gene and a second gene editing system targeting a gene selected from TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38.

[0422] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the TGFBR2 gene and a second gene editing system targeting a gene selected from FAS, CD7, CBLB, KLRC1, and CD38.

[0423] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the FAS gene and a second gene editing system targeting a gene selected from CD7, CBLB, KLRC1, and CD38.

[0424] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CD7 gene and a second gene editing system targeting a gene selected from CBLB and KLRC1.

[0425] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the CBLB gene and a second gene editing system targeting a gene selected from KLRC1 and CD38.

[0426] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting CD38.

[0427] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting CD38.

[0428] In some embodiments, the present disclosure provides a kit comprising a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting a gene selected from PD1, TGFBR2, CISH, CD38, CBLB, TIGIT, TIM-3, LAG3, FAS, and TGFBR2.

[0429] In some embodiments of the gene editing systems described herein, the guide RNA ((primary) single guide RNA and / or accessory guide RNA) is a dual RNA structure formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, the crRNA comprises a spacer sequence and is capable of forming a base pair structure with the tracrRNA, and wherein the base pair structure binds to the Cas protein. In some embodiments, the crRNA further comprises a linker sequence that comprises a protein-binding motif. For the purposes of the present disclosure, when the guide RNA is a dual RNA structure of crRNA and tracrRNA, the "CRISPR motif" refers to the base pair structure formed between the crRNA and the tracrRNA.

[0430] In some embodiments, the gene editing system is a LIGO-RNA-based gene editing system, as described in PCT / CN2023 / 096482, which is incorporated herein by reference in its entirety. Those skilled in the art will be able to design corresponding crRNA-tracrRNA pairs based on the sgRNA and hsgRNA disclosed herein.

[0431] In a LigoRNA-based gene editing system, at least one guide RNA is LigoRNA. The LigoRNA system comprises a dual RNA structure that can be used as a guide RNA in a CRISPR-based gene editing system. The dual RNA structure can be formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). For example, the LigoRNA system comprises an hgRNA set of hcrRNA and tracrRNA, and an mgRNA set of mcrRNA and tracrRNA. Preferably, all of these RNA molecules are no longer than 100 nucleotides.

[0432] Since the LigoRNA system consists of two short RNAs, it helps to solve the problem of synthesizing long single guide RNAs in previous gene editing systems. Chemically synthesized RNAs over 100 nt show much lower yields and purities, posing challenges to large-scale production and cost control.

[0433] The original types of crRNA and tracrRNA are capable of guiding nCas9-mediated DNA targeting. The crRNA and tracrRNA in the LigoRNA system are further modified. In some embodiments, MS2 or boxB hairpins are fused to the crRNA at multiple different sites. In some embodiments, at least one nucleotide in the crRNA and tracrRNA is modified, such as by 2'-O-methyl modification and / or 3'-thiolate modification.

[0434] In some embodiments, the crRNA comprises a spacer sequence and a linker sequence, wherein the linker sequence comprises at least one protein-binding motif, and the protein-binding motif is an RNA aptamer motif. In some embodiments, the protein-binding motif is selected from MS2, PP7, boxB, SfMu hairpin motif, telomerase Ku, and Sm7 binding motif, or variants thereof. An aptamer is a single-stranded oligonucleotide that folds into a defined structure and selectively binds a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells.

[0435] In some embodiments, the crRNA is capable of forming a base-paired structure with a trans-activating crRNA (tracrRNA). In some embodiments, the tracrRNA has the sequence of SEQ ID NO: 804 or 811.

[0436] In some embodiments, the crRNA comprises at least one nucleotide having a modification. In some embodiments, the modification is selected from 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halogen, 3'-thiophosphate, bridged nucleic acid (BNA), and locked nucleic acid (LNA). In some embodiments, the at least one nucleotide having a modification is any one of the first three nucleotides from the 3'-end of the engineered crRNA.

[0437] In some embodiments, the tracrRNA comprises at least one nucleotide having a modification. In some embodiments, the modification is selected from 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halogen, 3'-thiophosphate, bridged nucleic acid (BNA), and locked nucleic acid (LNA). In some embodiments, the at least one nucleotide having a modification is any one of the first three nucleotides from the 3'-end of the engineered tracrRNA.

[0438] In some embodiments, the crRNA and / or tracrRNA comprises at least one nucleotide having a modification. In some embodiments, the modification is selected from 2'-O-alkyl (e.g., 2'-O-methyl), 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halogen (e.g., 2'-fluoro), 3'-thiophosphate, bridged nucleic acid (BNA), and locked nucleic acid (LNA). In some embodiments, the crRNA and / or tracrRNA comprises nucleotides containing 2'-O-methyl and 3'-thiophosphate. In some embodiments, the first three nucleotides at the 5'-end of the crRNA and / or tracrRNA are modified with 2'-O-methyl and 3'-thiophosphate. In some embodiments, the first three nucleotides at the 3'-end of the crRNA and / or tracrRNA are modified with 2'-O-methyl, and the second to fourth nucleotides at the 3'-end of the crRNA and / or tracrRNA are modified with 3'-thiophosphate. In some embodiments, the first three nucleotides at the 5'-end of the crRNA and / or tracrRNA are modified with 2'-O-methyl and 3'-thiophosphate, and the first three nucleotides at the 3'-end of the crRNA and / or tracrRNA are modified with 2'-O-methyl, and the second to fourth nucleotides from the 3'-end of the crRNA and / or tracrRNA are modified with 3'-thiophosphate.

[0439] In some embodiments, the present disclosure provides a tBE system comprising two LigoRNA structures: an mcrRNA-tracrRNA base pairing structure and an hcrRNA-tracrRNA base pairing structure. In some embodiments, the mcrRNA comprises a boxB hairpin to generate an R-loop region for the desired base editing, and the hcrRNA comprises an MS2 hairpin to recruit a nucleotide deaminase (e.g., APOBEC linked to a nucleobase deaminase inhibitor (such as a cytosine deaminase inhibitor (dCDI)) domain) through a cleavage site (e.g., a TEV protease cleavage site). For example, to cleave the dCDI domain at the target site, TEVc fused to N22p is recruited by the boxB-containing mcrRNA and serves as a key component in the tBE system with free TEVn. In some embodiments, the mcrRNA and hcrRNA form base pairing structures with the same tracrRNA to localize the target DNA and cleave the dCDI domain at the target site to induce efficient base editing.

[0440] In some embodiments of the gene editing systems described herein, the gene editing system comprises

[0441] a. an hcrRNA comprising a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein-binding motif,

[0442] b. an mcrRNA comprising a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein-binding motif,

[0443] c. a first tracrRNA capable of forming a first base pair structure with the hcrRNA,

[0444] d. a second tracrRNA capable of forming a second base pair structure with the mcrRNA,

[0445] e. a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base pair structure,

[0446] f. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

[0447] g. a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to the first protein-binding motif,

[0448] Wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different.

[0449] In some embodiments of the gene editing system described herein, the gene editing system further comprises

[0450] a. A protease, or a polynucleotide encoding a protease, and

[0451] b. A nucleobase deaminase inhibitor domain,

[0452] wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain.

[0453] In some embodiments of the gene editing system described herein, the gene editing system comprises

[0454] a. An hcrRNA, which comprises a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein binding motif,

[0455] b. An mcrRNA, which comprises a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein binding motif,

[0456] c. A first tracrRNA, which is capable of forming a first base pair structure with the hcrRNA,

[0457] d. A second tracrRNA, which is capable of forming a second base pair structure with the mcrRNA,

[0458] e. A first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base pair structure,

[0459] f. A second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

[0460] g. A first fusion protein, which comprises a nucleobase deaminase or its catalytic domain and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA binding domain are optionally linked by a linker, and wherein the first RNA binding domain binds to the first protein binding motif,

[0461] h. A protease, or a polynucleotide encoding a protease,

[0462] i. a nucleobase deaminase inhibitor domain, and

[0463] j. a second fusion protein comprising a protease and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein,

[0464] wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different,

[0465] wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain,

[0466] wherein the protease and the second RNA-binding domain are optionally linked by a linker, and

[0467] wherein the second RNA-binding domain binds to a second protein-binding motif.

[0468] In some embodiments of the gene editing system described herein, the protease is split into a first protease fragment and a second protease fragment, wherein the first and / or second protease fragment alone cannot cleave the cleavage site.

[0469] In some embodiments of the gene editing system described herein, wherein the gene editing system comprises

[0470] a. an hcrRNA comprising a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein-binding motif,

[0471] b. an mcrRNA comprising a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein-binding motif,

[0472] c. a first tracrRNA capable of forming a first base pair structure with the hcrRNA,

[0473] d. a second tracrRNA capable of forming a second base pair structure with the mcrRNA,

[0474] e. a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base pair structure,

[0475] f. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

[0476] g. A first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to a first protein-binding motif,

[0477] h. A protease, or a polynucleotide encoding a protease, wherein the protease is split into a first protease fragment and a second protease fragment, and wherein the first and / or second protease fragment alone cannot cleave the cleavage site.

[0478] i. A nucleobase deaminase inhibitor domain,

[0479] j. A second fusion protein comprising the first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, and

[0480] k. A third fusion protein comprising the second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker,

[0481] wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different,

[0482] wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain,

[0483] wherein the mcrRNA further comprises a third protein-binding motif,

[0484] wherein the second RNA-binding domain binds to a second protein-binding motif, and

[0485] wherein the third RNA-binding domain binds to a third protein-binding motif.

[0486] In some embodiments of the gene editing system described herein, the gene editing system comprises

[0487] a. An hcrRNA comprising a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein-binding motif,

[0488] b. An mcrRNA comprising a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein-binding motif,

[0489] c. A first tracrRNA that is capable of forming a first base pair structure with the hcrRNA,

[0490] d. A second tracrRNA that is capable of forming a second base pair structure with the mcrRNA,

[0491] e. A first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base pair structure,

[0492] f. A second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

[0493] g. A first fusion protein that comprises a nucleobase deaminase or its catalytic domain and a first RNA-binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA-binding domain are optionally linked by a linker, and wherein the first RNA-binding domain binds to a first protein-binding motif,

[0494] h. A protease, or a polynucleotide encoding the protease, wherein the protease is split into a first protease fragment and a second protease fragment, and wherein the first and / or second protease fragment alone cannot cleave the cleavage site.

[0495] i. A nucleobase deaminase inhibitor domain,

[0496] j. A second fusion protein that comprises the first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA-binding domain are optionally linked by a linker, and

[0497] k. A third fusion protein that comprises the second protease fragment and a third RNA-binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA-binding domain are optionally linked by a linker,

[0498] wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different,

[0499] wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site of the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain,

[0500] wherein the mcrRNA further comprises a third protein-binding motif,

[0501] wherein the second RNA binding domain binds to a second protein binding motif,

[0502] wherein the third RNA binding domain binds to a third protein binding motif, and

[0503] wherein the second RNA binding domain and the third RNA binding domain are the same or different, and the second protein binding motif and the third protein binding motif are the same or different.

[0504] In some embodiments of the gene editing system described herein, the gene editing system comprises

[0505] a. an hcrRNA comprising a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein binding motif,

[0506] b. an mcrRNA comprising a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein binding motif,

[0507] c. a first tracrRNA capable of forming a first base pair structure with the hcrRNA,

[0508] d. a second tracrRNA capable of forming a second base pair structure with the mcrRNA,

[0509] e. a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base pair structure,

[0510] f. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

[0511] g. a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA binding domain are optionally linked by a linker, and wherein the first RNA binding domain binds to the first protein binding motif,

[0512] h. a protease, or a polynucleotide encoding the protease, wherein the protease is split into a first protease fragment and a second protease fragment, and wherein the first and / or second protease fragment alone cannot cleave the cleavage site.

[0513] i. a nucleobase deaminase inhibitor domain,

[0514] j. A second fusion protein comprising a first protease fragment and a second RNA-binding domain, or a polynucleotide encoding the second fusion protein,

[0515] wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different,

[0516] wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for a protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain,

[0517] wherein the first protease and the second RNA-binding domain are optionally linked by a linker, and

[0518] wherein the second RNA-binding domain binds to a second protein-binding motif.

[0519] polynucleotide

[0520] In another aspect, the present disclosure provides a polynucleotide encoding an hgRNA and / or an mgRNA disclosed in at least one of the gene editing systems herein.

[0521] In another aspect, the present disclosure provides a polynucleotide encoding all components of the gene editing system herein other than the first and second Cas proteins.

[0522] In another aspect, the present disclosure provides a polynucleotide encoding all components of the gene editing system herein.

[0523] In another aspect, the present disclosure provides a kit comprising a polynucleotide encoding all components of the gene editing system herein other than the first and second Cas proteins, and a polynucleotide encoding the first and / or second Cas protein of the gene editing system herein. In some embodiments, the first and second Cas proteins are the same Cas protein.

[0524] The polynucleotides disclosed herein can be obtained by methods known in the art. For example, polynucleotides can be obtained from cloned DNA (e.g., from a DNA library) by chemical synthesis, by cDNA cloning, or by cloning genomic DNA or fragments thereof purified from the desired cells. When producing polynucleotides recombinantly, any method known to those skilled in the art for identifying nucleic acids encoding the desired gene can be used. Any method available in the art can be used to obtain full-length (i.e., covering the entire coding region) cDNA or genomic DNA encoding the desired protein, e.g., from a cell or tissue source. Modified or variant polynucleotides can be engineered from wild-type polynucleotides using standard recombinant DNA methods. Any available method known in the art for cloning and isolating nucleic acid molecules can be used to clone or isolate polynucleotides. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.

[0525] Methods for amplifying polynucleotides can be used to isolate polynucleotides encoding the desired protein, including, for example, polymerase chain reaction (PCR) methods. PCR can be carried out using any method or procedure known in the art. Exemplary methods include using a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). Nucleic acids containing the gene of interest can be used as source material from which nucleic acid molecules encoding the desired polypeptide can be amplified. For example, DNA and mRNA preparations, cell extracts, tissue extracts from appropriate sources (e.g., testis, prostate, breast), fluid samples (e.g., blood, serum, saliva), samples from healthy and / or diseased subjects can be used in the amplification method. The source can be from any eukaryotic species, including but not limited to vertebrates, mammals, humans, pigs, cows, cats, birds, horses, dogs, and other primate sources. Nucleic acid libraries can also be used as source material. Primers can be designed to amplify the desired polynucleotide. For example, primers can be designed based on the expressed sequence that gives rise to the desired polynucleotide. Primers can be designed based on the reverse translation of the polypeptide amino acid sequence. Degenerate primers can be used for amplification if desired. Oligonucleotide primers that hybridize to sequences at the 3' and 5' ends of the desired sequence can be used as primers to amplify from a nucleic acid sample by PCR. Primers can be used to amplify the entire full-length polynucleotide or a truncated sequence thereof. The nucleic acid molecules produced by amplification can be sequenced and confirmed to encode the desired polypeptide.

[0526] vector

[0527] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding the hgRNA and / or mgRNA disclosed herein.

[0528] In another aspect, the present disclosure provides a vector comprising the polynucleotides disclosed herein.

[0529] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding all components of the gene editing system disclosed herein except the first and second Cas proteins.

[0530] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding all components of the gene editing system disclosed herein.

[0531] In some embodiments, the vector is a plasmid or viral vector.

[0532] In some embodiments, the vector is a polycistronic vector.

[0533] In another aspect, the present disclosure provides a kit comprising the vector disclosed above, and a vector comprising a polynucleotide encoding the first and / or second Cas protein of the gene editing system disclosed herein.

[0534] Any method known in the art for inserting a DNA fragment into a vector can be used to construct an expression vector comprising the polynucleotides disclosed herein. These methods can include in vitro recombinant DNA and synthetic techniques as well as in vivo (genetic) recombination. The polynucleotides disclosed herein can be operably linked to control sequences in the expression vector to ensure protein expression. Such control sequences can include, but are not limited to, a leader or signal sequence, a promoter (e.g., a native or heterologous promoter), a ribosome binding site, an enhancer or activator element, translation initiation and termination sequences, and transcription initiation and termination sequences, and control sequences are selected that are compatible with the host cell selected for expressing the protein. Constitutive or inducible promoters known in the art are also contemplated. The promoter can be a naturally occurring promoter, a hybrid promoter that combines elements of more than one promoter, or a synthetic promoter. The expression construct can be present on an episome (e.g., a plasmid) in the cell, or the expression construct can be inserted into a chromosome (e.g., at a locus). In some embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. In some embodiments, the vector is an expression vector comprising a nucleotide sequence encoding a variant polypeptide, the variant polypeptide being operably linked to at least one regulatory control sequence. Regulatory control sequences for use herein include promoters, enhancers, and other expression control elements. In some embodiments, the expression vector is designed for selection of the host cell to be transformed, the particular variant polypeptide to be expressed, the copy number of the vector, the ability to control that copy number, and / or the expression of any other protein encoded by the vector (e.g., an antibiotic marker).

[0535] Vectors can include, but are not limited to, viral vectors and plasmid DNA. Viral vectors can include, but are not limited to, adenoviral vectors, lentiviral vectors, retroviral vectors, and adeno-associated viral vectors. Typically, expression vectors contain selectable markers, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to allow detection of those cells transformed with the desired DNA sequence. Suitable vectors, promoters, and enhancer elements are known in the art; many are commercially available for generating the recombinant constructs described herein. In some embodiments, the vector is a polycistronic vector. In some embodiments, the vector is a bicistronic or tricistronic vector. Bicistronic or polycistronic expression vectors can include (1) multiple promoters fused to each open reading frame; (2) splicing signals inserted between genes; (3) fusion of genes, the expression of which is driven by a single promoter; and (4) a proteolytic cleavage site (self-cleaving peptide) inserted between genes or an internal ribosome entry site (IRES) inserted between genes.

[0536] Polycistronic vectors are used to co-express multiple genes in the same cell. Two strategies are most commonly used to construct polycistronic vectors. First, the internal ribosome entry site (IRES) element is commonly used in bicistronic vectors. The IRES element serves as another ribosome recruitment site, allowing translation to start from an internal region of the mRNA. Thus, two proteins are translated from one mRNA. The IRES element is quite large (usually 500 - 600 bp) (Pelletier et al., 1988; Jang et al., 1988). The engineered CD47 protein disclosed herein has a smaller size compared to the wild-type full-length human CD47 and can thus be used with the IRES element in polycistronic vectors with limited packaging capacity.

[0537] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding the hgRNA and / or mgRNA disclosed herein.

[0538] cell

[0539] In another aspect, the present disclosure provides a cell comprising the gene editing system disclosed herein.

[0540] In another aspect, the present disclosure provides a cell comprising the polynucleotide disclosed herein. In some embodiments, the cell further comprises a polynucleotide encoding all components of the gene editing system disclosed herein except the first and / or second Cas protein.

[0541] In another aspect, the present disclosure provides a cell comprising a vector disclosed herein. In some embodiments, the cell further comprises a polynucleotide encoding all components of the gene editing system disclosed herein other than the first and second Cas proteins.

[0542] In another aspect, the present disclosure provides a cell comprising a kit disclosed herein.

[0543] In some embodiments, the cell is a stem cell.

[0544] In some embodiments, the cell is a pluripotent stem cell. A pluripotent stem cell is a cell that has the ability to self-renew by division and develop into the three primary germ cell layers of an early embryo and, thus, into all the cells of the adult body, but not into extraembryonic tissues such as the placenta. Embryonic stem cells and induced pluripotent stem cells are pluripotent stem cells.

[0545] In some embodiments, the cell is an embryonic stem cell (ESC). An embryonic stem cell is a pluripotent stem cell derived from the inner cell mass of a blastocyst (early pre-implantation embryo).

[0546] In some embodiments, the cell is an induced pluripotent stem cell (iPSC). An iPSC is derived from adult somatic cells that have been genetically reprogrammed back to an embryo-like pluripotent state, which enables the development of an unlimited source of any type of cell required for therapeutic purposes.

[0547] As used herein, "pluripotent stem cells" have the potential to differentiate into any of the three germ layers: endoderm (such as the stomach wall, gastrointestinal tract, lung, etc.), mesoderm (such as muscle, bone, blood, urogenital tissues, etc.) or ectoderm (such as epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cells" also encompasses induced pluripotent stem cells (iPSC or iPS cells), or pluripotent stem cell types derived from non-pluripotent cells. In some embodiments, pluripotent stem cells are generated or produced from cells that are not pluripotent cells. In other words, pluripotent stem cells can be the direct or indirect progeny of non-pluripotent cells. Examples of parental cells include somatic cells that have been reprogrammed by various means to induce a pluripotent, undifferentiated phenotype. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods for inducing iPS cells are known in the art and are further described below. (See, e.g., Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009); each of which is incorporated herein by reference in its entirety). As used herein, "hiPSC" is a human induced pluripotent stem cell. In some embodiments, as used herein, "pluripotent stem cells" also encompasses mesenchymal stem cells (MSC) and / or embryonic stem cells (ESC).

[0548] In some embodiments, the cells are hematopoietic stem cells. Hematopoietic stem cells are pluripotent progenitor cells that can develop into all types of blood cells, including myeloid and lymphoid cells. The lymphoid lineage includes T cells, B cells, and natural killer (NK) cells. HSCs can be found in several organs, such as peripheral blood, bone marrow, and umbilical cord blood (Lee JY, Hong SH. Hematopoietic Stem Cells and Their Roles in Tissue Regeneration. Int J Stem Cells. 2020;13(1):1-12. doi:10.15283 / ijsc19127).

[0549] In some embodiments, the cells are immune cells. Immune cells refer to cells that participate in the functions of the immune system, including the innate immune system and the adaptive immune system. In some embodiments, the immune cells are selected from T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, monocytes, granulocytes, and mast cells.

[0550] Cell immunotherapy, also known as adoptive cell therapy, is an innovative treatment method that aims to utilize the body's immune system to eliminate cancer. To better identify and kill tumor cells, immune cells (including T cells, NK cells, γδ T cells, natural killer T (NKT) cells, and even macrophages) can be engineered to express antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) (Xie, Guozhu, et al. "CAR-NK cells: A promising cellular immunotherapy for cancer." EBioMedicine 59 (2020): 102975; Liu, Enli, et al. "Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors." New England Journal of Medicine 382.6 (2020): 545-553). A chimeric antigen receptor (CAR, also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is a receptor protein that has been engineered to give T cells the ability to target specific proteins.

[0551] In some embodiments, the cells are immune cells comprising a chimeric antigen receptor (CAR). In some embodiments, the cells are immune cells comprising a chimeric antigen receptor (CAR), wherein the immune cells are T cells, NK cells, γδ T cells, NKT cells, or macrophages.

[0552] In some embodiments, the cells are T cells. In some embodiments, the T cells comprise a chimeric antigen receptor (CAR). In some embodiments, the cells are CAR-T cells.

[0553] T cells are a type of lymphocyte. T cells are one of the white blood cells of the immune system and play a central role in the adaptive immune response. CAR-T cells are T cells that have been genetically engineered to produce an artificial chimeric antigen receptor. CAR-T cells can be CD4+ and CD8+, and a 1:1 ratio of the two cell types provides a synergistic anti-tumor effect. CAR-T cells can be derived from T cells in the patient's own blood (autologous) or from T cells of another healthy donor (allogeneic). T cells can be obtained from many sources, including but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from blood units collected from a subject using any number of techniques known to those of ordinary skill in the art, such as sedimentation, e.g., FICOLL TM separation, antibody-conjugated bead-based methods, e.g., MACS TM separation (Miltenyi).

[0554] In some embodiments, the cells are NK cells. In some embodiments, the NK cells comprise a chimeric antigen receptor (CAR). In some embodiments, the NK cells are CAR-NK cells.

[0555] NK cells, also known as large granular lymphocytes, are a type of cytotoxic lymphocyte. The role of NK cells is similar to that of cytotoxic T cells in the vertebrate adaptive immune response. CAR-NK cells are NK cells that have been genetically engineered to produce an artificial chimeric antigen receptor. Compared to CAR-T cells, CAR-NK cell infusion has a reduced risk of GvHD. In some embodiments, in addition to killing tumor target cells in a CAR-dependent manner, CAR-NK cells may also eliminate cancer cells in a CAR-independent manner. CAR-NK cells still possess natural cytotoxic activity against tumor cells (Oei, Vincent Yi Sheng, et al. "Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors Intrinsic Functionality of NK Cells Affects CAR Retargeting." Cancer immunology research 6.4 (2018): 467-480.).

[0556] In some embodiments, the cells are primary cells. Primary cells are isolated directly from human or animal tissues using enzymatic or mechanical methods. Once isolated, they are placed in an artificial environment in plastic or glass containers and provided with a special culture medium containing essential nutrients and growth factors to support proliferation. Primary cells can be of two types: adherent cells or suspension cells. Adherent cells require attachment for growth and are called anchorage-dependent cells. Adherent cells are typically derived from the tissues of organs. Suspension cells do not require attachment for growth and are called anchorage-independent cells. Most suspension cells are isolated from the blood system, but some tissue-derived cells can also be used in suspension, such as liver cells or intestinal cells. Although primary cells generally have a limited lifespan, they offer many advantages compared to cell lines. Primary cell culture enables researchers to study the donor, not just the cells. When establishing experimental models, various factors can be considered, such as age, medical history, race, and gender. With the growing trend of personalized medicine, this donor variability and tissue complexity can be achieved using primary cells, but it is difficult to replicate with cell lines that are inherently more systematic and homogeneous and cannot capture the true diversity of living tissues.

[0557] In some embodiments, the cells are differentiated cells. Differentiated cells are cells that have undergone differentiation. They are mature cells that perform special functions. Some examples of differentiated cells are epithelial cells, skin fibroblasts, endothelial cells lining blood vessels, smooth muscle cells, liver cells, nerve cells, human cardiomyocytes, etc. Generally, these cells have unique morphology, metabolic activity, membrane potential, and responsiveness to signals that promote their functions in body tissues or organs.

[0558] In some embodiments, the differentiated cells are differentiated from pluripotent stem cells. In some embodiments, the differentiated cells are differentiated from iPSCs or ESCs.

[0559] composition

[0560] In another aspect, the present disclosure provides a composition comprising the gene editing system disclosed herein.

[0561] In another aspect, the present disclosure provides a composition comprising the cells disclosed herein.

[0562] As used herein, the term "composition" includes, but is not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to an active agent formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals either alone or in combination with one or more other types of therapies. It should also be understood that, if desired, the compositions of the present disclosure can be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There are virtually no limitations on other components that can also be included in the composition, provided that the additional reagents do not adversely affect the ability of the composition to deliver the intended therapy. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0563] The composition may also contain a pharmaceutically acceptable carrier, diluent, or excipient. As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, surfactant, or emulsifying agent that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable fats; paraffin; silicone; bentonite; silicic acid; zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

[0564] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solutions, preferably physiological saline; Ringer's solution; isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides that can be used as solvents or suspension media; polyethylene glycol; glycerol; propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral products can be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0565] The composition can suitably be developed for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular or another route of administration.

[0566] treatment method

[0567] In another aspect, the present disclosure provides a method for reducing the immunogenicity of cells, comprising introducing any one or more of the gene editing systems disclosed herein into cells. By reducing the immunogenicity of cells administered to a subject, their expansion and persistence after administration can be enhanced.

[0568] In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, monocytes, granulocytes or mast cells. In some embodiments, the immune cells comprise chimeric antigen receptors. In some embodiments, the cells are T cells. In some embodiments, the T cells comprise chimeric antigen receptors. In some embodiments, the cells are CAR-T cells. In some embodiments, the cells are NK cells. In some embodiments, the NK cells comprise chimeric antigen receptors. In some embodiments, the NK cells are CAR-NK cells. In some embodiments, the cells are differentiated from pluripotent stem cells. In some embodiments, the cells are differentiated from iPSCs or ESCs. In some embodiments, the cells are primary cells.

[0569] In another aspect, the present disclosure provides a method for reducing the graft-versus-host (GvH) reaction associated with administering allogeneic cells to a subject, comprising reducing the immunogenicity of allogeneic cells by introducing any one or more of the gene editing systems disclosed herein into the allogeneic cells.

[0570] In some embodiments, the allogeneic cells are immune cells. In some embodiments, the immune cells are T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, monocytes, granulocytes, or mast cells. In some embodiments, the immune cells comprise chimeric antigen receptors. In some embodiments, the allogeneic cells are T cells. In some embodiments, the T cells comprise chimeric antigen receptors. In some embodiments, the cells are CAR-T cells. In some embodiments, the allogeneic cells are NK cells. In some embodiments, the NK cells comprise chimeric antigen receptors. In some embodiments, the NK cells are CAR-NK cells. In some embodiments, the cells are differentiated from pluripotent stem cells. In some embodiments, the cells are differentiated from iPSCs or ESCs. In some embodiments, the cells are primary cells.

[0571] One or more gene editing systems that can be used in a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction can be one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve gene editing systems selected from the following: gene systems targeting the PDCD1 gene, TRAC gene, B2M gene, CD52 gene, CTLA4 gene, TIGIT gene, TIM3 gene, LAG3 gene, CISH gene, TGFBR2 gene, FAS gene, CD7 gene, CBLB gene, KLRC1 gene, and CD38 gene disclosed herein.

[0572] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting the TRAC gene, B2M gene, CD52 gene, CTLA4 gene, TIGIT gene, TIM3 gene, LAG3 gene, CISH gene, TGFBR2 gene, FAS gene, and / or CD7 gene into the cells.

[0573] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TRAC gene and a second gene editing system targeting the PDCD1 gene, B2M gene, CD52 gene, CTLA4 gene, TIGIT gene, TIM3 gene, LAG3 gene, CISH gene, TGFBR2 gene, FAS gene, and / or CD7 gene into the cells.

[0574] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CD7 gene and a second gene editing system targeting the TRAC gene, B2M gene, CD52 gene, CTLA4 gene, TIGIT gene, TIM3 gene, LAG3 gene, CISH gene, TGFBR2 gene, FAS gene, and / or PDCD1 gene into a cell.

[0575] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TRAC gene and a second gene editing system targeting a gene selected from CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, and CD38 into a cell.

[0576] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CD52 gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0577] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the B2M gene and a second gene editing system targeting a gene selected from PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0578] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the PDCD1 gene and a second gene editing system targeting a gene selected from CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0579] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CTLA4 gene and a second gene editing system targeting a gene selected from TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0580] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TIGIT gene and a second gene editing system targeting a gene selected from TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0581] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TIM3 gene and a second gene editing system targeting a gene selected from LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0582] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the LAG3 gene and a second gene editing system targeting a gene selected from CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0583] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CISH gene and a second gene editing system targeting a gene selected from TGFBR2, FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0584] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing a graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TGFBR2 gene and a second gene editing system targeting a gene selected from FAS, CD7, CBLB, KLRC1, and CD38 into a cell.

[0585] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the FAS gene and a second gene editing system targeting a gene selected from CD7, CBLB, KLRC1, and CD38 into a cell.

[0586] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CD7 gene and a second gene editing system targeting a gene selected from CBLB and KLRC1 into a cell.

[0587] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the CBLB gene and a second gene editing system targeting a gene selected from KLRC1 and CD38 into a cell.

[0588] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting the CD38 gene into a cell.

[0589] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, which comprises introducing a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting the CD38 gene into a cell.

[0590] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the KLRC1 gene and a second gene editing system targeting a gene selected from PD1, TGFBR2, CISH, CD38, CBLB, TIGIT, TIM-3, LAG3, FAS, and TGFBR2 into a cell.

[0591] For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising knocking out two, three, or four of TRAC, CD52, B2M, PDCD1, CTLA4, TIGIT, TIM3, LAG3, CISH, TGFBR2, FAS, CD7, CBLB, KLRC1, and / or CD38 using the corresponding gene editing systems disclosed herein. For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity and / or reducing graft-versus-host (GvH) reaction, comprising introducing a first gene editing system targeting the TRAC gene, a second gene editing system targeting the CD52 gene, a third gene editing system targeting the PDCD1 gene, and a fourth gene editing system targeting the CD7 gene into a cell.

[0592] Table 12. mgRNA spacer and hgRNA spacer

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607] Table 36 mgRNA and hgRNA

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614] Table 37

[0615]

[0616]

[0617]

[0618] Examples

[0619] gene editing efficiency test

[0620] To apply the tBE system to generate stop codons or disrupt splice sites in target genes (TRAC gene, B2M gene, CD52 gene, PDCD1 gene, CTLA4 gene, TIGIT gene, TIM3 gene, LAG3 gene, CISH gene, TGFBR2 gene, FAS gene, CD7 gene, CBLB gene, KLRC1 gene, and CD38 gene), 15 pairs of mgRNA / hgRNA targeting the TRAC gene, 9 pairs of mgRNA / hgRNA targeting the CD52 gene, 45 pairs of mgRNA / hgRNA targeting the B2M gene, 47 pairs of mgRNA / hgRNA targeting PDCD1, 23 pairs of mgRNA / hgRNA targeting CTLA4, 47 pairs of mgRNA / hgRNA targeting TIGIT, 41 pairs of mgRNA / hgRNA targeting TIM3, 85 pairs of mgRNA / hgRNA targeting LAG3, 31 pairs of mgRNA / hgRNA targeting CISH, 19 pairs of mgRNA / hgRNA targeting TGFBR2, 26 pairs of mgRNA / hgRNA targeting FAS, 36 pairs of mgRNA / hgRNA targeting CD7, 18 pairs of mgRNA / hgRNA targeting CBLB, 9 pairs of mgRNA / hgRNA targeting KLRC1, and 19 pairs of mgRNA / hgRNA targeting CD38 were designed. The tBE system containing these respective guide RNA pairs was used to induce C-to-T base editing in codons CAA (Gln), CAG (Gln), TGG (Trp, C-to-T on the opposite strand), or CGA (Arg) in the target genes to generate stop codons TAA, TAG, or TGA. Genomic DNA was extracted 72 hours after plasmid transfection into cells. The C-to-T editing efficiency of different mgRNA / hgRNA pairs with tBE at the target sites was analyzed. Sanger sequencing results showed that tBE could perform efficient base editing to generate stop codons in the target genes.

[0621] The tBE system containing these guide RNA pairs was also used to induce G-to-A (C-to-T on the opposite strand) base editing in GT or AG splice sites to disrupt the GU-AG canonical splicing pattern. Sanger sequencing results showed that tBE also induced high base editing efficiency at these target sites.

[0622] The base editors, mgRNAs, hgRNAs, and base editing methods disclosed herein can be applied to perform highly specific and efficient base editing in the genomes of various eukaryotes.

[0623] plasmid construction

[0624] The primer set (hg-mg1&2-U1-TRAC_FOR / mg1-TRAC-Exon1-AG1_REV) was used to amplify the fragment hg-mg1&2-U1-TRAC-MS2 (operator in the hgRNA scaffold)-U6 (mgRNA promoter)-mg1-TRAC-Exon1-AG1 using the template pUC57-mgRNA-MS2-U6. The fragment hg-mg1&2-U1-TRAC-MS2-U6-mg1-TRAC-Exon1-AG1 was then ligated into BsmBI-linearized U6-ccdB-boxB-tBE-V5 to generate the vector ptBE-V5-TRAC-E1-AG1-U1. Other combinations with different on-target hgRNAs and mRNAs were constructed using the same strategy.

[0625] cell culture and transfection

[0626] 293FT cells were maintained in DMEM + 10% FBS and were regularly tested to exclude mycoplasma contamination. For base editing with variant BE, 293FT cells were seeded in 24-well plates at a density of 1×10 5 / well and transfected with 250 μl of serum-free Opti-MEM containing 2.5 μl of LIPOFECTAMINE LTX, 1 μl of LIPOFECTAMINE plus, 0.5 μg of the tBE-V5 expression vector, and 0.5 μg of the pEFS-nSpCas9 or pEFS-nSpCas9-NG expression vector. After 24 hours, puromycin was added to the medium at a final concentration of 4 μg ml -1 . After another 48 hours, genomic DNA was extracted from the cells using QuickExtractT DNA Extraction Solution for subsequent sequencing analysis. The target genomic sequence was PCR amplified using the high-fidelity DNA polymerase PrimeSTAR HS, with primer sets flanking the detected mgRNA target site.

[0627] The base substitution frequency at each target site was calculated by EditR analysis. See http: / / baseeditr.com / .

[0628] For obtaining Figure 2-5The base substitution calculations, statistical analyses, and other related steps for the data shown are substantially the same as those disclosed in the "Methods" section of Wang, Lijie, et al., Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations, Nature Cell Biology 23.5 (2021): 552-563, the content of which is incorporated herein by reference in its entirety.

[0629] The gene editing results obtained from the above experiments are exemplified in Figure 2-5 below.

[0630] Verification at the protein level

[0631] The results of tBE editing were further verified at the protein level using flow cytometry or western blotting. mgRNA / hgRNA pairs that showed relatively higher editing efficiency for the TRAC, B2M, CD52, PDCD1, CISH, TGFBR2, FAS, CBLB, KLRC1, or CD38 genes were tested, either individually for each gene or in combinations of two to three genes. The results confirmed that C-to-T or G-to-A base editing induced by tBE can disrupt the protein expression of the target gene, whether single-strand, double-strand, or triple-strand editing (singlex, duplex or triplex editing).

[0632] Although the present disclosure has been specifically shown and described with reference to specific embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[0633] The sequences of SEQ ID NO: 828-920 are as follows:

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

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[0646]

[0647]

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[0649]

[0650]

[0651]

Claims

1. A gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the T cell receptor alpha constant (TRAC) gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 1-5.

2. The gene editing system according to claim 1, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

3. The gene editing system according to claim 1, wherein the nucleic acid sequences of said mgRNA and hgRNA respectively comprise:

4. A gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the CD52 gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 6-8.

5. The gene editing system according to claim 4, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

6. The gene editing system according to claim 4, wherein the nucleic acid sequences of said mgRNA and hgRNA respectively comprise:

7. A gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the beta-2 microglobulin (B2M) gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 9-19.

8. The gene editing system according to claim 7, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

9. The gene editing system according to claim 7, wherein the nucleic acid sequences of said mgRNA and hgRNA respectively comprise:

10. A gene editing system comprising a guide RNA (mgRNA) and a helper guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the programmed cell death protein 1 (PDCD1) gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 20-38.

11. The gene editing system according to claim 10, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

12. The gene editing system according to claim 10, wherein the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

13. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytotoxic T lymphocyte-associated protein 4 (CTLA4) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 247-256.

14. The gene editing system according to claim 14, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

15. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the T cell immunoreceptor with Ig and ITIM domains (TIGIT) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 278-294.

16. The gene editing system according to claim 15, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

17. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the hepatitis A virus cellular receptor 2 (HAVCR2 / TIM3) gene, the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 323-337.

18. The gene editing system according to claim 17, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

19. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the lymphocyte activation 3 (LAG3) gene, and the hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 364-396.

20. The gene editing system according to claim 19, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

21. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the cytokine-inducible SH2-containing protein (CISH) gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 472-482.

22. The gene editing system according to claim 21, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

23. The gene editing system according to claim 21, wherein the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

24. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the transforming growth factor beta receptor 2 (TGFBR2) gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 504-510.

25. The gene editing system according to claim 24, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

26. The gene editing system according to claim 24, wherein the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

27. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding the mgRNA and / or hgRNA, wherein the mgRNA comprises an mgRNA spacer targeting the Fas cell surface death receptor (FAS) gene, and the hgRNA comprises an hgRNA spacer, and wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 530-541.

28. The gene editing system according to claim 27, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer respectively comprise:

29. The gene editing system according to claim 27, wherein the nucleic acid sequences of the mgRNA and the hgRNA respectively comprise:

30. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the CD7 gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 565-575.

31. The gene editing system according to claim 30, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

32. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding mgRNA and / or hgRNA, wherein mgRNA comprises an mgRNA spacer targeting the Cbl proto-oncogene B (CBLB) gene, and hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 609-618.

33. The gene editing system according to claim 32, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

34. The gene editing system according to claim 32, wherein the nucleic acid sequences of said mgRNA and hgRNA respectively comprise:

35. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding said mgRNA and / or hgRNA, wherein said mgRNA comprises an mgRNA spacer targeting the killer cell lectin-like receptor C1 (KLRC1) gene, and said hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of said mgRNA spacer comprises a sequence selected from SEQ ID NO: 637-641.

36. The gene editing system according to claim 35, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

37. The gene editing system according to claim 35, wherein the nucleic acid sequences of said mgRNA and hgRNA respectively comprise:

38. A gene editing system comprising a guide RNA (mgRNA) and an auxiliary guide RNA (hgRNA), or at least one DNA polynucleotide encoding mgRNA and / or hgRNA, wherein mgRNA comprises an mgRNA spacer targeting the CD38 gene, hgRNA comprises an hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NO: 651-659.

39. The gene editing system according to claim 38, wherein the nucleic acid sequences of said mgRNA spacer and hgRNA spacer respectively comprise:

40. The gene editing system according to claim 38, wherein the nucleic acid sequences of the mgRNA and hgRNA respectively comprise:

41. The gene editing system according to any one of claims 1-40, comprising a. an hgRNA comprising a first CRISPR motif, an hgRNA spacer, and a first protein binding motif, or a DNA polynucleotide encoding the hgRNA, b. an mgRNA comprising a second CRISPR motif and an mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, c. a first CRISPR-associated protein (Cas protein), or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, d. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, e. a first fusion protein comprising a nucleobase deaminase or its catalytic domain and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or its catalytic domain and the first RNA binding domain are optionally linked by a linker, and wherein the first RNA binding domain binds to the first protein binding motif. Wherein the first Cas protein and the second Cas protein are the same or different.

42. The gene editing system according to claim 41, further comprising f. a protease, or a polynucleotide encoding the protease, and g. a nucleobase deaminase inhibitor domain, Wherein the nucleobase deaminase inhibitor domain is optionally linked to the nucleobase deaminase or its catalytic domain in the first fusion protein by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or its catalytic domain.

43. The gene editing system according to claim 42, further comprising a second fusion protein comprising a protease and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, Wherein the protease and the second RNA binding domain are optionally linked by a linker, Wherein the mgRNA further comprises a second protein binding motif, and Wherein the second RNA binding domain binds to the second protein binding motif.

44. The gene editing system according to claim 42, wherein the protease is split into a first protease fragment and a second protease fragment, and wherein the first and / or second protease fragment alone cannot cleave the cleavage site.

45. The gene editing system according to claim 44, further comprising h. a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally linked by a linker, and i. A third fusion protein comprising a second protease fragment and a third RNA-binding domain, or a polynucleotide encoding said third fusion protein, wherein said second protease fragment and said third RNA-binding domain are optionally linked by a linker, wherein said mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein said second RNA-binding domain binds to said second protein-binding motif, and wherein said third RNA-binding domain binds to said third protein-binding motif.

46. The gene editing system according to claim 45, wherein said second and third RNA-binding domains are the same or different, and said second and third protein-binding motifs are the same or different.

47. The gene editing system according to claim 44, further comprising a second fusion protein comprising said first protease fragment and a second RNA-binding domain, or a polynucleotide encoding said second fusion protein, wherein said first protease fragment and said second RNA-binding domain are optionally linked by a linker, wherein said mgRNA further comprises a second protein-binding motif, and wherein said second RNA-binding domain binds to said second protein-binding motif.

48. The gene editing system according to any one of claims 42-47, wherein said protease is TEV protease, TuMV protease, PPV protease, PVY protease, ZIKV protease or WNV protease.

49. The gene editing system according to claim 48, wherein said protease is TEV protease comprising the sequence of SEQ ID NO:

124.

50. The gene editing system according to claim 49, wherein said first TEV protease fragment comprises the sequence of SEQ ID NO:

125.

51. The gene editing system according to any one of claims 42-50, wherein said nucleobase deaminase inhibitor is an inhibitory domain of a nucleobase deaminase.

52. The gene editing system according to any one of claims 42-51, wherein said nucleobase deaminase inhibitor is an inhibitory domain of a cytidine deaminase.

53. The gene editing system according to claim 52, wherein said inhibitory domain of the cytidine deaminase comprises the amino acid sequence of SEQ ID NO:141 or SEQ ID NO:

142.

54. The gene editing system according to any one of claims 41-53, wherein the nucleotide deaminase of said first fusion protein is a cytidine deaminase.

55. The gene editing system according to claim 54, wherein said cytidine deaminase is selected from APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), APOBECI (Al), APOBEC3 (A3), APOBEC2 (A2), APOBEC4 (A4) and AICDA (AID).

56. The gene editing system according to claim 54, wherein the cytidine deaminase is a human or murine cytidine deaminase.

57. The gene editing system according to claim 56, wherein the catalytic domain of the cytidine deaminase is murine A3 cytidine deaminase domain 1 (mA3-CDAl) or human A3B cytidine deaminase domain 2 (hA3B-CDA2).

58. The gene editing system according to any one of claims 41-57, wherein the first fusion protein further comprises a uracil glycosylase inhibitor (UGI).

59. The gene editing system according to any one of claims 41-58, wherein the Cas protein is Cas9, inactivated Cas9 (dCas9) or Cas9 nickase (nCas9) selected from the following: SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl, LbCpfl, FnCpfl, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, AsCpfl, FnCpfl, SsCpfl, PcCpfl, BpCpfl, CmtCpfl, LiCpfl, PmCpfl, Pb3310Cpfl, Pb4417Cpfl, BsCpfl, EeCpfl, BhCasl2b, AkCasl2b, EbCasl2b, LsCasl2b, RfCasl3d, LwaCasl3a, PspCasl3b, PguCasl3b and RanCasl3b.

60. The gene editing system according to any one of claims 41-59, wherein the first protein-binding RNA motif and the first RNA-binding domain, the second protein-binding RNA motif and the second RNA-binding domain, and the third protein-binding RNA motif and the third RNA-binding domain are each independently selected from the MS2 bacteriophage operator stem-loop and the MS2 coat protein (MCP) or its RNA-binding segment, BoxB and N22P or its RNA-binding segment, telomerase Ku-binding motif and Ku protein or its RNA-binding segment, telomerase Sm7-binding motif and Sm7 protein or its RNA-binding segment, PP7 bacteriophage operator stem-loop and PP7 coat protein (PCP) or its RNA-binding segment, SfMu bacteriophage Com stem-loop and Com RNA-binding protein or its RNA-binding segment, and non-natural RNA aptamer and the corresponding aptamer ligand or its RNA-binding segment.

61. A polynucleotide encoding the mgRNA and / or hgRNA according to at least one of claims 1-40.

62. A polynucleotide encoding all components of the gene editing system according to any one of claims 41-60 except the first and second Cas proteins.

63. A kit comprising the polynucleotide according to claim 62, and a polynucleotide encoding the first and / or second Cas protein according to any one of claims 41-60.

64. A vector comprising the polynucleotide according to claim 61.

65. A vector comprising the polynucleotide according to claim 62.

66. The vector according to any one of claims 64-65, wherein the vector is a plasmid or a viral vector.

67. The vector according to any one of claims 64-66, wherein the vector is a polycistronic vector.

68. A kit comprising j. The vector according to any one of claims 64-67, k. A vector comprising a polynucleotide encoding the first and / or second Cas protein according to any one of claims 41-60.

69. A cell comprising the gene editing system according to any one of claims 1-60.

70. A cell comprising the polynucleotide according to any one of claims 61-62.

71. The cell according to claim 70, which further comprises a polynucleotide encoding the first and / or second Cas protein according to any one of claims 41-60.

72. A cell comprising the vector according to any one of claims 64-67.

73. The cell according to claim 72, which further comprises a vector comprising a polynucleotide encoding the first and / or second Cas protein according to any one of claims 41-60.

74. The cell according to any one of claims 69-73, wherein the cell is a stem cell.

75. The cell according to claim 74, wherein the stem cell is a pluripotent stem cell or a hematopoietic stem cell.

76. The cell according to claim 75, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or an embryonic stem cell.

77. The cell according to any one of claims 69-73, wherein the cell is an immune cell.

78. The cell according to claim 77, wherein the cell is selected from T cells, B cells, natural killer cells (NK cells), macrophages, dendritic cells, monocytes, granulocytes, and mast cells.

79. The cell according to any one of claims 59-73, wherein the cell is a T cell.

80. The cell according to claim 79, wherein the T cell comprises a chimeric antigen receptor (CAR).

81. The cell according to claim 80, wherein the T cell is a CAR-T cell.

82. The cell according to any one of claims 69-73, wherein the cell is a natural killer cell (NK cell).

83. The cell according to claim 82, wherein the NK cell comprises a chimeric antigen receptor.

84. The cell according to claim 83, wherein the NK cell is a CAR-NK cell.

85. A cell according to any one of claims 69 - 84, wherein the cell is a primary cell.

86. A cell according to any one of claims 69 - 73 and 77 - 84, wherein the cell is a differentiated cell.

87. A cell according to claim 86, wherein the cell is differentiated from a pluripotent stem cell.

88. A cell according to claim 87, wherein the pluripotent stem cell is an iPSC or an ESC.

89. A composition comprising a gene editing system according to any one of claims 1 - 60.

90. A composition comprising a cell according to any one of claims 69 - 88.

91. A kit comprising a first gene editing system according to any one of claims 1 - 3, and a second gene editing system according to any one of claims 4 - 34 and 38 - 40.

92. A kit comprising a first gene editing system according to any one of claims 4 - 6, and a second gene editing system according to any one of claims 10 - 40.

93. A kit comprising a first gene editing system according to any one of claims 7 - 9, and a second gene editing system according to any one of claims 10 - 40.

94. A kit comprising a first gene editing system according to any one of claims 10 - 12, and a second gene editing system according to any one of claims 13 - 40.

95. A kit comprising a first gene editing system according to any one of claims 13 - 14, and a second gene editing system according to any one of claims 15 - 40.

96. A kit comprising a first gene editing system according to any one of claims 15 - 16, and a second gene editing system according to any one of claims 17 - 40.

97. A kit comprising a first gene editing system according to any one of claims 17 - 18, and a second gene editing system according to any one of claims 19 - 40.

98. A kit comprising a first gene editing system according to any one of claims 19 - 20, and a second gene editing system according to any one of claims 21 - 40.

99. A kit comprising a first gene editing system according to any one of claims 21 - 23, and a second gene editing system according to any one of claims 24 - 40.

100. A kit comprising a first gene editing system according to any one of claims 25 - 26, and a second gene editing system according to any one of claims 27 - 40.

101. A kit comprising a first gene editing system according to any one of claims 27 - 29, and a second gene editing system according to any one of claims 30 - 40.

102. A kit comprising a first gene editing system according to any one of claims 30 - 31, and a second gene editing system according to any one of claims 32 - 37.

103. A kit, comprising the first gene editing system according to any one of claims 32-34, and the second gene editing system according to any one of claims 35-40.

104. A kit, comprising the first gene editing system according to any one of claims 35-37, and the second gene editing system according to any one of claims 38-40.

105. A kit, comprising at least one gene editing system, wherein each said gene editing system is according to any one of claims 1-40.

106. A method for reducing the immunogenicity of a cell, comprising introducing the gene editing system according to any one of claims 1-60 into said cell.

107. A method for reducing the graft-versus-host (GvH) reaction associated with administering allogeneic cells to a subject, comprising reducing the immunogenicity of said allogeneic cells by introducing the gene editing system according to any one of claims 1-60 into the allogeneic cells.

108. The method according to claim 107, wherein said allogeneic cells are immune cells.

109. The method according to claim 108, wherein said immune cells are T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, monocytes, granulocytes or mast cells.

110. The method according to claim 109, wherein said immune cells are T cells.

111. The method according to claim 110, wherein said T cells comprise a chimeric antigen receptor (CAR).

112. The method according to claim 111, wherein said T cells are CAR-T cells.

113. The method according to claim 109, wherein said immune cells are NK cells.

114. The method according to claim 113, wherein said NK cells comprise a chimeric antigen receptor (CAR).

115. The method according to claim 114, wherein said NK cells are CAR-NK cells.

116. The method according to claim 110 or 115, wherein said cells are differentiated from pluripotent stem cells.

117. The method according to claim 116, wherein said pluripotent stem cells are iPSCs or ESCs.

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