Compositions and methods for immunotherapy

Abstract

To provide methods for editing, e.g., altering a DNA sequence, within the TRBC1, TRBC2 and / or TRAC genes, e.g., methods for immunotherapy.SOLUTION: Provided is a method of altering a DNA sequence within the TRBC1 and / or TRBC2 gene, comprising delivering a composition to a cell, the composition comprising: (a) a guide RNA comprising a specific sequence; or (b) a nucleic acid encoding a guide RNA of (a); and optionally (c) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA- guided DNA binding agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 746,522, filed October 16, 2018. No. 62 / 747,037, filed October 17, 2018, and The benefit of prior art is claimed, and these U.S. provisional patent applications are incorporated herein by reference. It shall be deemed to have been used.

[0002] This application contains a sequence listing, submitted electronically in ASCII format, in its entirety. Created October 14, 2019. The SCII copy is named Sequence_Listing.txt and contains the The size is 272 kilobytes. [Background technology]

[0003] CRISPR (clustered regularly interspaced short palindromic repeats) red Regularly Interspaced Short Palindro mic Repeats)) are adaptive immune responses in bacteria to combat viral attacks. After exposure to the virus, short segments of viral DNA are transferred to the bacterial genome. The CRISPR locus containing the viral sequence is integrated into the CRISPR locus of the virus. This RNA, which contains a sequence complementary to the viral genome, is transcribed from the Ca It mediates targeting of the Cas9 protein to sequences within the viral genome. Proteins restrict viral targets by causing cleavage.

[0004] Recently, the CRISPR / Cas system has been applied to genome editing in eukaryotic cells. The introduction of specific single-strand breaks (SSBs) or double-strand breaks (DSBs) is mediated by non-homologous end joining (NSE). This allows for targeted sequence mutation via NHEJ or homology-directed repair (HDR).

[0005] Eyquem et al. (2017), Nature, Vol. 543 (No. 7643): pp. 113- 117 reported the development of a chimeric antigen receptor (CAR) TRAC using the CRISPR-Cas9 system. Targeting genes to specific loci has been reported to enhance tumor rejection. Moreover, such targeting to the TRAC locus inhibits tonic CAR signaling. and prevent the transfer of CAR to the CAR-like cells, and ensure effective internalization and elimination of CAR after single or repeated exposure to antigen. The differentiation and exhaustion of effector T cells is delayed due to the establishment of T cell proliferation and re-expression. However, existing approaches generate T cells that lack the desired activity against the target of interest. (CAR may be expressed from the TRAC locus, interact with CAR, and cause undesirable The purpose of this study was to generate T cells expressing endogenous TRBC gene products that could give rise to highly reactive T cells. (including, but not limited to, the use of immunotherapy). Goodness is required.

[0006] To this end, the following embodiments are provided. In some embodiments, the present invention provides a Using RNA-guided DNA binding agents such as CRISPR / Cas systems , substantially reducing or knocking out the expression of the TRBC gene and / or the TRAC gene. By targeting the native α subunit of the T cell receptor (also known as TCR) and / or provides compositions and methods that substantially reduce or eliminate the production of β subunits . Summary of the Invention

[0007] The invention described herein provides a method for the treatment of cancer by engineered T cell therapy (e.g., immunotherapy). The present invention relates to compositions and methods for the treatment of cancer, e.g., genomic Cells modified at a specific target sequence (containing a gRNA molecule targeting said target sequence) (including cells modified by the introduction of CRISPR systems), as well as the methods for producing them. For example, the present disclosure relates to methods for the treatment of cells, e.g., T cells, e.g., For example, T cells engineered to lack endogenous T cell receptor expression, e.g. ... T cells suitable for further manipulation to insert sequences, such as modified T cell receptors or mutant T cells. genome editing of T cells, which are further engineered to express T cell receptors such as mitochondrial receptors (TCRs) gRNA molecules, CRISPR systems, cells, and methods useful for immunotherapy and methods, which are provided by the present disclosure.

[0008] In a first aspect, the present invention provides a gRNA molecule comprising a tracrRNA and a crRNA. wherein the crRNA comprises a targeting domain that binds to a target sequence in a T cell. In one embodiment, the targeting domain is a T The T cells can be either autologous or allogeneic T cells. The T cells may be CD3 + T cells, CD4 + T cells and / or CD8 + T thin While the present invention is as provided in the claims, further embodiments are possible. The form is shown below.

[0009] The present invention provides a method for modifying the DNA sequence within the TRBC1 gene and / or the TRBC2 gene. A method of delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0010] The present invention further provides a method for modifying a DNA sequence, comprising the steps of: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i. Table 1 and / or for SEQ ID NOs: 1-89 and 179-184 is 15 consecutive nucleotides ±10 of any of the genomic coordinates listed in Table 3 a sequence containing nucleotides; ii. At least 17, at least 18, at least 19 of the sequences from (i). , or at least 20 consecutive nucleotides; iii. at least 99%, at least 98% for a sequence selected from (i); At least 97%, at least 96%, at least 95%, at least 94%, at least have at least 93%, at least 92%, at least 91%, or at least 90% identity Guide sequence; (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0011] The present invention further provides a method for reducing the expression of the TRBC1 gene and / or the TRBC2 gene. A method for causing delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 24; and v) a guide sequence comprising any one of SEQ ID NOs: 1 to 6; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0012] The present invention further provides a method of immunotherapy comprising: administering the composition to the subject, their autologous cells, and / or allogeneic cells; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 24; and v) a guide sequence comprising any one of SEQ ID NOs: 1 to 6; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0013] The present invention also provides a method for modifying a DNA sequence within a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i) a sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv) any one of SEQ ID NOs: 90 to 113 and 213 to 218 a guide sequence comprising: v) a guide sequence comprising any one of SEQ ID NOs: 90 to 95; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0014] The present invention further provides a method for reducing expression of a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i) a sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv) A guide including any one of SEQ ID NOs: 90 to 113 and 213 to 218. and v) a guide sequence comprising any one of SEQ ID NOs: 90 to 95; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid The present invention provides a method comprising:

[0015] The present invention also provides a method of immunotherapy comprising the steps of: administering the composition to a subject, their autologous cells, and / or allogeneic cells; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i) a sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv) any one of SEQ ID NOs: 90 to 113 and 213 to 218 a guide sequence comprising: v) a guide sequence comprising any one of SEQ ID NOs: 90 to 95; or (b) a nucleic acid encoding the guide RNA of (a.); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0016] The present invention further provides a method for modifying a DNA sequence, comprising the steps of: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from the following: i. SEQ ID NOs: 90-218, as described in either Table 2 and / or Table 3 A sequence containing 15 consecutive nucleotides ± 10 nucleotides of the selected genome coordinate column; ii. At least 17, at least 18, at least 19 of the sequences from (i). , or at least 20 consecutive nucleotides; iii. at least 99%, at least 98% for a sequence selected from (i); At least 97%, at least 96%, at least 95%, at least 94%, at least have at least 93%, at least 92%, at least 91%, or at least 90% identity Guide sequence; (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; acid, The present invention provides a method comprising:

[0017] The present invention further relates to a method for producing the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. 1. A method for modifying a DNA sequence in a gene, comprising: A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and delivering a nucleic acid encoding an A-binding agent or an RNA-guided DNA-binding agent. , The first guide RNA is i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 24; and v) a guide sequence comprising any one of SEQ ID NOs: 1 to 6; and The second guide RNA is i. A sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. Any one of SEQ ID NOs: 90 to 113 and 213 to 218 a guide sequence comprising: v. A guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method includes the step of:

[0018] The present invention further relates to a method for producing the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. 1. A method for reducing expression of a gene, comprising: A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and delivering a nucleic acid encoding an A-binding agent or an RNA-guided DNA-binding agent. , The first guide RNA i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 24; and v) a guide sequence comprising any one of SEQ ID NOs: 1 to 6; and a sequence selected from The second guide RNA is i) a sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii) SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv) any one of SEQ ID NOs: 90 to 113 and 213 to 218 a guide sequence comprising: v) a guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method includes the step of:

[0019] The invention further includes administering the composition to a subject, their autologous cells, or allogeneic cells. 1. A method of immunotherapy comprising: The composition comprises a first guide RNA, a second guide RNA, and optionally an RNA guide and a nucleic acid encoding a DNA-binding agent or an RNA-guided DNA-binding agent, The first guide RNA is i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 24; and v) a guide sequence comprising any one of SEQ ID NOs: 1 to 6; and The second guide RNA is i. A sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 Selected guide sequence; ii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. Any one of SEQ ID NOs: 90 to 113 and 213 to 218 a guide sequence comprising: v. A guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method includes the step of:

[0020] Without wishing to be bound by theory, it is believed that TRAC / TRBC double mutants (e.g. The use of TCRs (e.g., knockout mutants) can improve the proportion of engineered / transduced TCRs that reach the cell surface. This is advantageous in some embodiments within the scope of the present invention because it provides a larger capacity.

[0021] In some embodiments, TRAC knock-in of an exogenous TCR in an engineered cell is performed. TRBC knockout in combination with ATP resulted in greater activity in engineered cells. This is advantageous in obtaining native TCRs.

[0022] In some embodiments, TRAC knock-in of an exogenous TCR in an engineered cell is performed. TRBC knockout combined with cloning resulted in greater selectivity in engineered cells. This is advantageous when obtaining exogenous TCR.

[0023] Additionally, in some embodiments, the exogenous TCR in the engineered cells may be expressed as a TRAC. TRBC knockout combined with knock-in was measured by caspase assays etc. This may be advantageous in achieving greater cell death in some cases.

[0024] In some embodiments, TRAC knock-in of an exogenous TCR in an engineered cell is performed. TRBC knockout in combination with steroids can be achieved by administering inducible peptides (e.g., Wilms tumor gene expression). To achieve greater IFN-γ secretion in response to the WT1 antigen, is advantageous.

[0025] Furthermore, in some embodiments, the TRAC / TRBC double nodal receptors using the above TCRs are Blockout is effective in achieving greater IFN-γ secretion, e.g., in CD4+ cells. It is profitable.

[0026] Additionally, in some embodiments, the interferon response (IFNγ and and / or TNFα) to prevent transcription of the inserted donor construct from occurring in the endogenous TRAC. It is increased in cells that have undergone editing, driven by the promoter.

[0027] Additionally, in some embodiments, TRAC / TRB C double knockout achieves greater IFN-γ secretion in CD4+ cells Without wishing to be bound by theory, in some embodiments , CD, which is not usually strongly associated with reactivity to peptides presented on HLA class I. Such activity is rarely observed in 4+ cells.

[0028] Additionally, in some embodiments, TRAC / TRB The C double knockout exhibited a greater response in CD4+ cells, including in degranulation assays. Again, without wishing to be bound by theory, However, in some embodiments, reactivity to peptides presented on HLA class I is Such activity has not been observed in CD4+ cells, which do not normally bind strongly to This is it.

[0029] Additionally, in some embodiments, the TRAC / TRBC double knockout mutant is When tested in mixed lymphocyte culture reactions, etc., the TRAC single knockout mutant In comparison, it is advantageous in achieving a lesser degree of alloreactivity.

[0030] The present invention further provides a method for the expression of a heterologous immune receptor via intralocus insertion at the TRAC locus. A method for expressing a gene comprising the steps of: A cell is administered a first guide RNA, a second guide RNA, and an RNA-guided DNA-binding agent or delivering a nucleic acid encoding an RNA-guided DNA-binding agent; The first guide RNA is i) a guide sequence selected from SEQ ID NOs: 1 to 89; ii) at least 17 or at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii) at least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv) a guide sequence comprising any one of SEQ ID NOs: 1, 2, 3, 5, and 6; and v) a guide sequence comprising any one of SEQ ID NOs: 2, 3, 5, and 6; and The second guide RNA is i) a guide selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218 Do array; ii) a sequence selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218; At least 17, at least 18, at least 19, or at least 20 consecutive nucleotides; iii) selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218 At least 99%, at least 98%, at least 97%, at least At least 96%, at least 95%, at least 94%, at least 93%, at least 92% , a guide sequence with at least 91%, or at least 90% identity; iv) containing any one of SEQ ID NOs: 90, 95, 97, 185, and 214; Guide sequence; v) a guide sequence comprising any one of SEQ ID NOs: 90, 95, and 185; and vi) a guide sequence comprising SEQ ID NO: 90 or 214; The method includes the step of:

[0031] "Knockout" of the endogenous TRAC gene by a single cutting and / or editing event A further advantage is that if a template is introduced or provided during the editing process, In this case, the foreign polypeptide of interest can be inserted into the target locus of the editing event. , SEQ ID NOs: 90, 95, 97, 185, 203, 204, 205, 206, 210, 21 1, and / or 214. These guide RNAs A is introduced together with gRNAs targeting one or more loci of the TRBC gene. It is also possible.

[0032] The first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 90 may include:

[0033] The first guide RNA comprises the sequence of SEQ ID NO: 180, and the second guide RNA comprises the sequence of SEQ ID NO: 186 It may comprise the sequence:

[0034] The first guide RNA comprises any one of SEQ ID NOs: 1, 2, 3, 5, and 6, and the second guide RNA comprises any one of SEQ ID NOs: 1, 2, 3, 5, and 6. The idRNA may comprise the sequence of SEQ ID NO:90.

[0035] The first guide RNA comprises any one of SEQ ID NOs: 1, 2, 3, 5, and 6, and the second guide RNA comprises any one of SEQ ID NOs: 1, 2, 3, 5, and 6. The idRNA may comprise the sequence of SEQ ID NO:214.

[0036] First guide RNA, second guide RNA, and RNA-guided DNA binder or RNase The nucleic acid encoding the A-guided DNA binder may be introduced or administered substantially simultaneously.

[0037] DNA sequences within the TRBC1, TRBC2, and / or TRAC genes can be simultaneously modified.

[0038] The RNA-guided DNA-binding agent or a nucleic acid encoding the RNA-guided DNA-binding agent may be It may be introduced or administered as part of a modification method.

[0039] The above method is (a) TRBC1 gene, TRBC2 gene, and and / or introducing a double-strand break (DSB) within the TRAC gene; or (b) TRBC1 gene, TRBC2 gene, and and / or introducing a single-strand break (SSB) into the TRAC gene; or (c) TRBC1 gene, TRBC2 gene, and / or decreasing the expression of the TRAC gene; It may further include:

[0040] The method may further comprise introducing a nucleic acid sequence encoding a polypeptide of interest. However, if desired, (a) the one or more polypeptides of interest include a receptor; (b) the one or more polypeptides of interest comprise an immune receptor; (c) the one or more polypeptides of interest include a T cell receptor, and optionally , the above T cell receptor recognizes cancer antigens; (d) the one or more polypeptides of interest contain a WT1-specific T cell receptor; The T cell receptor recognizes WT1 or a fragment thereof; (e) the one or more polypeptides of interest include a chimeric antigen receptor, and further, optionally Thus, the chimeric antigen receptor recognizes a cancer antigen; or (f) The one or more polypeptides of interest include a WT1-specific chimeric antigen receptor. The chimeric antigen receptor recognizes WT1 or a fragment thereof.

[0041] The method may further include: a. Introducing TCR α chain and TCR β chain; b. introducing one or more nucleic acid sequences encoding the TCR alpha chain and the TCR beta chain; ; c. Introducing WT1-specific TCR α and β chains; d. Derivation of one or more nucleic acid sequences encoding the α and β chains of a WT1-specific TCR. Entering; e. Introducing a first TCR sequence selected from: (i) SEQ ID NO: 501; or SEQ ID NO:504; (ii) at least 99% of SEQ ID NO:501 or SEQ ID NO:504 %, at least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or at least 60% identity; and (ii) i) at least 20, at least 30, at least 10, or at least 20 copies of SEQ ID NO: 501 or SEQ ID NO: 504 At least 40, at least 50, at least 60, at least 70, at least 8 0, at least 90, at least 100, at least 150, at least 200 or a contiguous subsequence of at least 250 amino acids, and, introducing a second TCR sequence selected from: (i) SEQ ID NO: 502 or sequence No. 505; (ii) at least 99% to SEQ ID NO: 502 or SEQ ID NO: 505; At least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and iii) at least 20, at least 30 of SEQ ID NO: 502 or SEQ ID NO: 505; At least 40, at least 50, at least 60, at least 70, at least At least 80, at least 90, at least 100, at least 150, at least 2 a contiguous subsequence of at least 0, at least 250, or at least 300 amino acids , f. Introducing a first TCR sequence selected from: (i) SEQ ID NO: 501; or SEQ ID NO:513; (ii) at least 99% of SEQ ID NO:510 or SEQ ID NO:513 %, at least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and (iii) at least 20, at least 30 of SEQ ID NO: 510 or SEQ ID NO: 513 pcs, at least 40 pcs, at least 50 pcs, at least 60 pcs, at least 70 pcs, At least 80, at least 90, at least 100, at least 150, at least a contiguous subsequence of at least 200 or at least 250 amino acids, and, introducing a second TCR sequence selected from: (i) SEQ ID NO: 511 or sequence No. 514; (ii) at least 99% to SEQ ID NO: 511 or SEQ ID NO: 514; At least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and iii) at least 20, at least 30 of SEQ ID NO: 511 or SEQ ID NO: 514; At least 40, at least 50, at least 60, at least 70, at least At least 80, at least 90, at least 100, at least 150, at least 2 a contiguous subsequence of at least 0, at least 250, or at least 300 amino acids , g. Introducing a nucleic acid sequence comprising a sequence encoding the first TCR sequence of (e) or (f). thing; h. Introducing a nucleic acid sequence comprising a sequence encoding the second TCR sequence of (e) to (f). ; i. introducing a nucleic acid sequence comprising the nucleic acid sequences of (g) and (h); j. SEQ ID NO: 500, 503, 506, 509, 512, 515, 518, or 5 21, or at least 99%, at least 9 5%, or at least 90% identity to the target amino acid sequence, by introducing a nucleic acid sequence encoding the same; k. A TCR α chain polypeptide and a TCR selected from the following (i) to (viii): β-chain polypeptide, or at least 99%, at least 95%, or at least Introduce an amino acid sequence that has 90% identity with: i) SEQ ID NO: 501 and SEQ ID NO: 502; ii) SEQ ID NO: 504 and SEQ ID NO: 505; iii) SEQ ID NO: 507 and SEQ ID NO: 508; iv) SEQ ID NO: 510 and SEQ ID NO: 511; v) SEQ ID NO: 513 and SEQ ID NO: 514; vi) SEQ ID NO: 516 and SEQ ID NO: 517; vii) SEQ ID NO: 519 and SEQ ID NO: 520; l. Nucleic acids encoding the TCR α chain polypeptide and the TCR β chain polypeptide of (k). Introducing arrays.

[0042] The first nucleic acid sequence may be flanked by sequences homologous to the first target locus. The sequence may be flanked by sequences homologous to a second target locus. At least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least The sequence may be at least 35, or at least 40 nucleotides in length.

[0043] The first target locus is the TRAC gene, the TRBC1 gene, or the TRBC2 gene. The second target locus may be, for example, the TRAC gene. The TRBC1 gene or the TRBC2 gene may be used. For example, the TRBC1 gene or TRBC2 gene.

[0044] The introduced nucleic acid sequence, or the first nucleic acid sequence and the second nucleic acid sequence, may contain a promoter region or the like. The nucleic acid may be "promoterless"; .

[0045] The introduced nucleic acid sequence, or the first and second nucleic acid sequences, are functionally linked to a promoter. Optionally, the promoter may be linked to an EF-1α promoter (sequence Column number 603).

[0046] The introduced nucleic acid sequence, or the first and second nucleic acid sequences, may be a vector, a transfer vector, may be introduced via transfection, lipid nanoparticles, or microinjection .

[0047] The vector may be a viral vector, and further, if desired, the viral The vector is an adeno-associated virus vector.

[0048] The present invention also provides a method for inserting a TCR (such as a WT1-specific TCR) into a gene locus, comprising: Guide arrangement selected from row numbers 90, 95, 97, 98, 185, 214, and 218 a first guide RNA for inserting a TCR comprising a sequence, and optionally, (i) an RNA Nucleic acids encoding guided DNA binding agents or RNA-guided DNA binding agents; and / or or (ii) a donor nucleic acid molecule encoding a TCR (e.g., a WT1-specific TCR). The method further comprises obtaining a sequence selected from SEQ ID NOs: 1 to 89. The method further comprises delivering a second guide RNA comprising SEQ ID NOs: 179-1. 84.

[0049] The TCR may be a WT1-specific TCR, including: i) SEQ ID NO: 500, 503, 506, 509, 512, 515, 518, or 5 21, or at least 99%, at least 9 5%, an amino acid sequence with at least 90% identity; or ii) A TCR α chain polypeptide and a TCR selected from the following (i) to (viii): Rβ chain polypeptide, or at least 99%, at least 95%, or at least Amino acid sequences with at least 90% identity: i) SEQ ID NO: 502 and SEQ ID NO: 503; ii) SEQ ID NO: 504 and SEQ ID NO: 505; iii) SEQ ID NO: 507 and SEQ ID NO: 508; iv) SEQ ID NO: 510 and SEQ ID NO: 511; v) SEQ ID NO: 513 and SEQ ID NO: 514; vi) SEQ ID NO: 516 and SEQ ID NO: 517; vii) SEQ ID NO: 519 and SEQ ID NO: 520; viii) SEQ ID NO: 522 and SEQ ID NO: 523.

[0050] The present invention also provides a composition comprising: a. A guide RNA comprising: i. a guide sequence selected from SEQ ID NOs: 1-89; or ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; or iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% a guide sequence with identity; or iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; or v. a guide sequence comprising any one of SEQ ID NOs: 1 to 6; and, optionally, b. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent .

[0051] The composition contains D in the TRBC1 gene and / or TRBC2 gene in cells. The composition can be used to modify the TRBC1 gene and the NA sequence in cells. The composition may be used to induce the expression of the TRBC2 gene and / or the TRBC2 gene. The RNA may contain a sequence selected from any one of SEQ ID NOs: 196 to 200. .

[0052] The present invention also provides a composition comprising: a. A guide RNA comprising: i. A sequence selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218 the selected guide sequence; or ii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; or iii. SEQ ID NOs: 90 to 178, 185, and 213 to 218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; and teeth iv. Any one of SEQ ID NOs: 90 to 113 and 213 to 218 containing a guide sequence; or v. a guide sequence comprising any one of SEQ ID NOs: 90-95; and, optionally, the law of nature b. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent .

[0053] The composition is used to modify a DNA sequence within a TRAC gene in a cell. The composition may be used to reduce the expression of the TRAC gene in a cell. The guide RNAs may be those represented by SEQ ID NOs: 185 to 192 and SEQ ID NO: 20. It can be selected from any of 1 to 212.

[0054] The present invention also provides a cell modified by the methods described herein. may be modified ex vivo.

[0055] The cells can be T cells. The cells can be CD3 + T cells, CD4 + T cells, and and / or CD8 + The cell may be a mammalian cell, a primate cell, or a human T cell. It may be a cell.

[0056] The cells may lack endogenous T cell receptors. These cells may be suitable for generating T cells expressing the CA cell receptor. It can be used to generate T cells that express R.

[0057] Cells modified as described herein may be CD3 - The cell may be a cell. , before modification it was CD3 + It may be a cell.

[0058] The cells may further comprise one or more nucleic acid sequences encoding a polypeptide of interest. However, if desired, (a) the one or more polypeptides of interest include a receptor; (b) the one or more polypeptides of interest comprise an immune receptor; (c) the one or more polypeptides of interest include a T cell receptor, and optionally , wherein the T cell receptor is specific for WT1; or (d) the one or more polypeptides of interest include a chimeric antigen receptor, and further, optionally Therefore, the chimeric antigen receptor is specific for WT1.

[0059] The cells are infected with one or more nucleic acid sequences encoding the α and β chains of the exogenous T cell receptor. The cells may further comprise sequences encoding the gamma and delta chains of the exogenous T cell receptor. Alternatively, it may further comprise multiple nucleic acid sequences.

[0060] One or more genes encoding the α and β chains of the foreign T cell receptor of the cells described herein. The nucleic acid sequence can be within the TRAC locus of the genome. The one or more nucleic acid sequences encoding the gamma and delta chains of the foreign T cell receptor of The gene may be within the TRAC locus.

[0061] The sequence of the TCR alpha chain is (i) SEQ ID NO: 501 or SEQ ID NO: 504; (ii) the sequence At least 99%, at least 95%, at least 100% of SEQ ID NO: 501 or SEQ ID NO: 504 At least 90%, at least 85%, at least 80%, at least 70%, or (of) a sequence having at least 60% identity; and SEQ ID NO: 501 or SEQ ID NO: 504 At least 20, at least 30, at least 40, at least 50, At least 60, at least 70, at least 80, at least 90, at least 10 0, at least 150, at least 200, or at least 250 amino acids a contiguous subsequence of: The sequence of the TCR β chain is (i) SEQ ID NO: 502 or SEQ ID NO: 505; (ii) the sequence At least 99%, at least 95%, at least 100% of SEQ ID NO: 502 or SEQ ID NO: 505 At least 90%, at least 85%, at least 80%, at least 70%, or (of) a sequence having at least 60% identity; and SEQ ID NO: 502 or SEQ ID NO: 505 At least 20, at least 30, at least 40, at least 50, At least 60, at least 70, at least 80, at least 90, at least 10 0, at least 150, at least 200, at least 250, or at least The amino acid sequence may be selected from a continuous subsequence of up to 300 amino acids.

[0062] The TCR alpha chain is set forth in any of SEQ ID NOs: 500, 501, 503, and 504. The β TCR chain may be encoded by a nucleic acid sequence of SEQ ID NO: 500, 502, , 503, and 505.

[0063] The sequence of the TCR α chain is: (i) SEQ ID NO: 513; (ii) a sequence slightly different from SEQ ID NO: 513; at least 99%, at least 95%, at least 90%, at least 85%, at least sequences with 80%, at least 70%, or (of) at least 60% identity; and and at least 20, at least 30, at least 40, or at least 100 copies of SEQ ID NO: 513. At least 50, at least 60, at least 70, at least 80, at least 9 0, at least 100, at least 150, at least 200, at least 25 0, at least 300, at least 350, at least 400, at least 50 0, at least 600, at least 700, or at least 800 amino acids and a contiguous subsequence of The sequence of the TCR β chain is (i) SEQ ID NO: 514; (ii) a sequence slightly different from SEQ ID NO: 514 at least 99%, at least 95%, at least 90%, at least 85%, at least sequences with 80%, at least 70%, or (of) at least 60% identity; and and at least 20, at least 30, at least 40, or at least 100 copies of SEQ ID NO: 514. At least 50, at least 60, at least 70, at least 80, at least 9 0, at least 100, at least 150, at least 200, at least 25 0, at least 300, at least 350, at least 400, at least 50 0, at least 600, at least 700, or at least 800 amino acids A contiguous subsequence of

[0064] The alpha TCR chain can be SEQ ID NO: 513 and the TCR beta chain is SEQ ID NO: 514. obtain.

[0065] One or more genes of the cells disclosed herein are expressed from an endogenous promoter. It is possible.

[0066] One or more genes of the cells disclosed herein are expressed from a heterologous promoter. Optionally, the heterologous promoter is the EF-1α promoter.

[0067] The modified cells contain genes encoding the α and β chains of the foreign T cell receptor, and and / or may contain genes encoding the gamma and delta chains of an exogenous T cell receptor. Alpha, beta, gamma, and delta chains are known in the art. See No. 2018 / 197492.

[0068] The α and β chains of the exogenous T cell receptor may be present in the TRAC locus of the genome. The α and β chains of the native T cell receptor are provided within the transcript, and P2A or other cleavage sequences are are separated by

[0069] The present invention also provides a cell population comprising the cells disclosed herein, wherein the modified population comprises about 5% or more of the cells. More than 0%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, More than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% CD3 - In cells A cell population is provided.

[0070] The present invention also provides a cell population comprising the cells disclosed herein, wherein the modified population comprises approximately 50% or more of the cells. More than %, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, about More than 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% endogenous T cell receptors The body is deficient, providing a cell population.

[0071] The present invention also provides a cell population comprising the cells disclosed herein, wherein the population contains a TR The expression of the BC1 gene, the TRBC2 gene, and / or the TRAC gene is At least about 50%, at least about 55%, at least about 60% compared to a single cell population , at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% depleted.

[0072] The decreased expression may be a decreased expression of the TRBC1 gene. The decreased expression may be a decrease in the expression of the TRAC gene. .

[0073] 10 to 100% of the cell population, for example, 30 to 99% of the population, expressing the TRBC1 gene, T They may have indels in the RBC2 gene and / or the TRAC gene.

[0074] Furthermore, 30-35%, 35-40%, 40-45%, 45-50%, 50% of the population ~55%, 55~60%, 60~65%, 65~70%, 70~75%, 75~80%, 80-85%, 85-90%, 90-95%, or 95-99% of cases involve the TRBC1 gene. , TRBC2 gene, and / or TRAC gene.

[0075] The indel or insertion may be in the TRBC1 gene. The indel or insertion may be in the TRBC2 gene. It may be in the child.

[0076] The methods and compositions for use described herein may comprise the step of: and / or editing of the TRBC2 gene. The compositions for use as described herein may result in editing of the TRAC gene. The method or composition for use described herein may comprise the steps of: This may result in editing.

[0077] Editing can be calculated as the proportion of the population that has undergone editing (edit rate or indel rate). The concentration rate is 30-35%, 35-40%, 40-45%, 45-50%, 50-55% of the population. %, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80- It can be 85%, 85-90%, 90-95%, or 95-99%.

[0078] The compositions described herein may include an sgRNA comprising: (e) any one of SEQ ID NOs: 179 to 184 and 196 to 200; or (f) a guide sequence selected from any one of SEQ ID NOs: 1 to 89; or (g) a guide sequence selected from SEQ ID NOs: 1 to 24; or (h) a guide sequence selected from SEQ ID NOs: 1 to 6.

[0079] The compositions described herein may include an sgRNA comprising: (i) any one of SEQ ID NOs: 186 to 192 and 201 to 212; or (j) any of SEQ ID NOs: 90 to 178, 185, and 213 to 218 a guide sequence selected from one of the following: (k) a guide sequence selected from SEQ ID NOs: 90 to 113 and 213 to 218 ;or (l) a guide sequence selected from SEQ ID NOs: 90 to 95.

[0080] The target sequence is a sequence of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. , can be in the first exon, second exon, third exon, or fourth exon. The sequences may be selected from the human TRBC1 gene, the human TRBC2 gene, and / or the human TRAC gene. It can be present in genes.

[0081] The target sequence is a sequence of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. The target sequence may be present in the first exon of the TRBC1 gene, the TRBC2 gene, and The target sequence may be located in the second exon of the TRBC1 gene and / or the TRAC gene. The nucleotide sequence may be present in the third exon of the TRBC2 gene, the TRBC2 gene, and / or the TRAC gene. The target sequence is a sequence of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. It can be present in the fourth exon.

[0082] The guide sequence is selected from the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. The guide sequence may be complementary to a target sequence in the positive strand of the TRBC1 gene. , TRBC2 gene, and / or TRAC gene, and target sequences in the minus strand. and may be complementary.

[0083] The first guide sequence is the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. The composition may be complementary to a first target sequence in the positive strand of the molecule, and the composition may be a TRBC. 1 gene, TRBC2 gene, and / or a second target within the minus strand of the TRAC gene It further comprises a second guide sequence complementary to the sequence.

[0084] The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178, And may further comprise the nucleotide sequence of SEQ ID NO: 400, The nucleotide sequence follows the 3' end of the guide sequence.

[0085] The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178, And may further comprise the nucleotide sequence of SEQ ID NO: 401, The nucleotide sequence follows the 3' end of the guide sequence.

[0086] The guide RNA may be modified according to the pattern of SEQ ID NO: 300, where N is collectively any one of the guide sequences of SEQ ID NOs: 1 to 89. Each N can be any natural or non-natural nucleotide, and the N is a guide sequence. The guide sequences guide Cas9 to the TRBC1 gene, the TRBC2 gene, and and / or targeting the TRAC gene.

[0087] The first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 90 may include:

[0088] Any of the above methods may further include: (a) introducing a first nucleic acid sequence comprising a nucleic acid sequence selected from: (i) SEQ ID NO: (ii) at least 99%, at least 95%, at least at least 90%, at least 85%, at least 80%, at least 70%, or (of (iii) a sequence having at least 60% identity to SEQ ID NO: 250; and At least 20, at least 30, at least 40, at least 50, at least 60 pieces, at least 70 pieces, at least 80 pieces, at least 90 pieces, at least 100 pieces, a few pieces At least 150, at least 200, at least 250, at least 300, At least 350, at least 400, at least 500, at least 600, a contiguous subsequence of at least 700 or at least 800 nucleotides, and, introducing a second nucleic acid sequence comprising a nucleic acid sequence selected from: (i) SEQ ID NO: 25; 2; (ii) at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, or (of) less and (iii) a sequence having at least 2 sequences identical to SEQ ID NO: 252. 0, at least 30, at least 40, at least 50, at least 60, a few At least 70, at least 80, at least 90, at least 100, at least At least 150, at least 200, at least 250, at least 300, at least At least 350, at least 400, at least 500, at least 600, at least at least 700, at least 800, or at least 900 consecutive nucleotides Subarray, (b) introducing a first nucleic acid sequence comprising a nucleic acid sequence selected from: (i) SEQ ID NO: (ii) at least 99%, at least 95%, at least 100% to SEQ ID NO: 513; at least 90%, at least 85%, at least 80%, at least 70%, or (of (iii) a sequence having at least 60% identity to SEQ ID NO: 513; and At least 20, at least 30, at least 40, at least 50, at least 60 pieces, at least 70 pieces, at least 80 pieces, at least 90 pieces, at least 100 pieces, a few pieces At least 150, at least 200, at least 250, at least 300, At least 350, at least 400, at least 500, at least 600, a contiguous subsequence of at least 700 or at least 800 nucleotides, and, introducing a second nucleic acid sequence comprising a nucleic acid sequence selected from: (i) SEQ ID NO: 51 4; (ii) at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, or (of) less and (iii) a sequence having at least 2 sequences identical to SEQ ID NO: 514. 0, at least 30, at least 40, at least 50, at least 60, a few At least 70, at least 80, at least 90, at least 100, at least At least 150, at least 200, at least 250, at least 300, at least At least 350, at least 400, at least 500, at least 600, at least at least 700, at least 800, or at least 900 consecutive nucleotides Subarray, (c) introducing a nucleic acid sequence comprising the first nucleic acid sequence of (a) and the second nucleic acid sequence of (a); that; or (d) introducing a nucleic acid sequence comprising the first nucleic acid sequence of (b) and the second nucleic acid sequence of (b); thing.

[0089] The sgRNA has at least 99% affinity to a sequence selected from SEQ ID NOs: 1 to 89, and at least at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 % identity to the guide sequence.

[0090] Guide RNA can be chemically synthesized. Guide RNA can be contained in RNPs. The template nucleic acid may be contained in a viral vector. The template nucleic acid may be non-viral. Suitable viral vectors may be those contained in viral delivery constructs. These are well known in the art and include, for example, retroviruses, adenoviruses, lentiviruses, and Viral vectors include viruses, adeno-associated viruses, and hybrids thereof. The vectors are lentiviral (LV) or adeno-associated viral (AAV) vectors. It is possible.

[0091] The guide RNA may optionally include one or more of the following modifications: i) 2'-O-methyl (2'-O-Me) modified nucleotides; ii) phosphorothioate (PS) internucleotide bonds; iii) 2'-fluoro (2'-F) modified nucleotides; iv) Modifications in one or more of the first five nucleotides at the 5' end of the guide RNA Decoration, v) Modification of one or more of the last five nucleotides at the 3' end of the guide RNA , vi) a PS bond between the first four nucleotides at the 5' end of the guide RNA; vii) a PS bond between the last four nucleotides at the 3' end of the guide RNA; viii) 2'-OM in the first three nucleotides of the 5' end of the guide RNA e modified nucleotides, ix) 2'-O-Me modification in the last three nucleotides of the 3' end of the guide RNA Decorative nucleotides.

[0092] The guide RNA may comprise the nucleotides of SEQ ID NO: 300 with the modifications described above.

[0093] The composition may further comprise a pharmaceutically acceptable excipient.

[0094] The LNPs can include a biodegradable ionic lipid, for example, the ionic lipid is (9Z,1 2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((( 3-(Diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca -9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl) oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl The compound is (9Z,12Z)-propyl octadeca-9,12-dienoate.

[0095] The LNP can include a neutral lipid, for example, the neutral lipid is DSPC.

[0096] The LNP can include a helper lipid, for example, the helper lipid is cholesterol.

[0097] The LNPs can include stealth lipids, e.g., stealth lipids. The lipid is PEG2k-DMG.

[0098] The composition may further comprise an RNA-guided DNA binding agent. The composition of the A-guided DNA binder is contained in a ribonucleoprotein (RNP). obtain.

[0099] The composition may comprise an RNA-guided DNA binding agent, such as Cas9, or an RNA-guided The RNA-guided DNA binding agent may comprise an mRNA encoding the DNA binding agent, e.g. , which may be encoded by the Cas9 gene, or the Cas9 protein may be.

[0100] The composition may be a pharmaceutical formulation and may further comprise a pharmaceutically acceptable carrier.

[0101] The invention also relates to the use of a composition, formulation, population, or cell described herein in the preparation of a medicament. The present invention provides for the use of vesicles.

[0102] The present invention also relates to the use of a composition, formulation, population, or cell described herein in the treatment of cancer. The present invention provides for the use of vesicles.

[0103] The present invention also relates to the use of the compositions, formulations, populations, or compositions described herein in the immunotherapy of a subject. provides for the use of cells.

[0104] The present invention also relates to the use of the present invention in the treatment of tumors that overexpress Wilms tumor antigen (WT1). Uses of the compositions, preparations, populations, or cells described herein are provided.

[0105] The present invention also relates to the compositions, formulations, and compositions described herein for use in the treatment of a disease or disorder. A population or cell is provided.

[0106] The present invention also relates to compositions, formulations, populations, or compositions described herein for use in immunotherapy. Provide the cells.

[0107] The present invention also relates to compositions, formulations, populations, or compositions described herein for use in the treatment of cancer. provides the cells.

[0108] The present invention also provides therapeutic use in the treatment of tumors that overexpress the Wilms tumor antigen (WT1). The present invention provides a composition, preparation, population, or cell described herein.

[0109] The present invention also provides a method for treating a disease in a human or animal comprising administering the compositions, formulations, populations, or cells described herein. and providing a composition, formulation, population, or cell described herein in a method of treating a mammal or animal. do.

[0110] The present invention also provides a method for treating a disease in a human or animal comprising administering the compositions, formulations, populations, or cells described herein. The use of the compositions, formulations, populations, or compositions described herein in a method for treating cancer in a human or animal. provides the cells.

[0111] The present invention also provides a method for treating a disease in a human or animal comprising administering the compositions, formulations, populations, or cells described herein. the compositions, preparations, populations, or cells described herein in methods of immunotherapy of a mammal or animal. to provide.

[0112] The present invention also provides a method for treating a disease in a human or animal comprising administering the compositions, formulations, populations, or cells described herein. and a method for treating tumors overexpressing Wilms' tumor antigen (WT1) in animals. The present invention provides a composition, preparation, population, or cell described herein.

[0113] The guide RNA may have a sequence selected from any of SEQ ID NOs: 1-89.

[0114] Guide RNAs are SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence may be selected from any of the 18.

[0115] In addition, the guide sequence is modified to 20% or more, 30% or more, 40% or more, or 50% or more. Disclosed embodiments are selected from a group of guide sequences that provide the frequency of indels in the variant. will be done.

[0116] In addition, the guide sequence is modified to 20% or more, 30% or more, 40% or more, or 50% or more. A guide sequence selected from a group of sequences that results in a frequency of insertion of the donor nucleic acid molecule in the mutant. An embodiment is disclosed.

[0117] Also, the composition of any of the above embodiments for the preparation of a medicament for treating a subject; The use of the preparation, population, or cell is disclosed. The subject may be a human or an animal (e.g., a human). In some embodiments, the subject is a human.

[0118] Also, any one of the TRBC1 gene, the TRBC2 gene, and the TRAC gene used in the treatment of, or the modification (e.g., indications therein) of, one or more formation of a gene encoding a nucleotide sequence, or the formation of a frameshift or nonsense mutation therein Disclosed are any of the above compositions or formulations for use in

[0119] In any of the above aspects and embodiments of the cell, the targeting of the first gRNA molecule a gene containing a target sequence complementary to the targeting domain, and optionally a second gRN a gene containing a target sequence complementary to the targeting domain of the A molecule, and / or is a gene containing a target sequence complementary to the targeting domain of the third gRNA molecule. is a gene containing a target sequence complementary to the targeting domain of the first gRNA molecule and, optionally, the targeting domain of a second gRNA molecule. a gene containing a target sequence complementary to the target of the third gRNA molecule; The functional product of a gene containing a target sequence complementary to the binding domain is reduced or eliminated. It has been modified to reflect this.

[0120] In another aspect, the present invention provides a method of providing immunotherapy in a subject, the method comprising administering to a subject a therapeutically effective amount of a compound as described herein. An effective amount of the cells described, e.g., cells of any of the cell aspects and embodiments described above, is administered to a subject. The method includes administering to

[0121] In each embodiment of the above method, the method further comprises administering a lymphocyte depleting or immunosuppressive agent to the subject after the administration of the lymphocyte depleting or immunosuppressive agent. a cell as described herein, such as a cell of any of the cell aspects and embodiments described above, In another aspect, the present invention provides a method for treating a cell (e.g., a cell population) comprising administering an effective amount to a subject. ) is provided.

[0122] Immunotherapy is the treatment of disease by activating or suppressing the immune system. Immunotherapies designed to induce or amplify cell proliferation are classified as activating immunotherapies. Lymphocyte-based immunotherapy has been shown to be effective in treating several cancers. , macrophages, dendritic cells, natural killer cells (NK cells), and / or cells Immune effector cells, such as cytotoxic T lymphocytes (CTLs), are expressed on the surface of tumor cells. They can be programmed to react to abnormal antigens that have been detected, thus Immunotherapy involves using components of the immune system to destroy tumors or other cancerous cells.

[0123] In another aspect, the invention is a method for producing cells (e.g., cell populations) for immunotherapy. For example, by (a) introducing into the cell a gRNA molecule (a gRNA molecule described herein); By introducing two or more gRNA molecules as disclosed herein, T cell receptor by reducing or eliminating the expression of one or more or all components of the TCR and (b) modifying the cells; and (b) propagating the cells. The cells of the present invention are provided, for example, by expressing polypeptides that mediate TCR / CD3 ζ chain signaling. The vector is suitable for further modification by the introduction of a heterologous sequence encoding a peptide. In some embodiments, the polypeptide is a wild-type TCR or a variant TCR. Cells may also be engineered to bind to other antigens, e.g., by the introduction of heterologous sequences encoding other antigen-binding moieties. (sexual) T cell receptors, e.g., chimeric antigens engineered to target specific proteins The recombinant vector may be suitable for further modification by the introduction of heterologous sequences encoding a CAR receptor (CAR). CARs are available as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors. is also known.

[0124] In another aspect, the present invention provides a method of treating a subject in need thereof, comprising administering to a subject a therapeutically effective amount of a compound as described herein. Aspects and embodiments of the methods of producing the cells described above, e.g. administering cells (e.g., cell populations) produced by any of the methods, A method is provided. [Brief explanation of the drawings]

[0125] [Figure 1] 1 shows the extent of TRAC editing in HEK-Cas9 cells. [Figure 2] 1 shows the extent of TRAC editing in human CD3+ T cells. [Figure 3] 1 shows the amount of TCR expression after TRAC editing in human CD3+ T cells. [Figure 4] Figure 4 shows the extent of TRBC editing in HEK-Cas9 cells. Figure 4A shows editing in TRBC1, and Figure 4B shows editing in TRBC2. [Figure 5] 1 shows the extent of TRBC editing in human CD3+ T cells. [Figure 6] 1 shows the expression level of TCR in human CD3+ T cells after TRBC editing. [Figure 7] Biochemical off-target analysis (measurement of potential off-target editing sites) is shown for the TRBC guide, TRAC guide, and control guide (sequence number 193 and sequence number 194). [Figure 8] Figure 8 shows the generation of cells, including TCR-edited T cells, by knocking out the T cell receptor and inserting a TCR construct. Figure 8A shows the efficiency of T cell receptor knockout. Figure 8B shows the efficiency of lentiviral transfection of the TCR construct. Figure 8C shows the expression of the TCR insert in edited T cells. Figure 8D shows the phenotype of TCR-edited T cells. [Figure 9] The ability of T cells with TCR insertions to kill primary AML blasts is shown. Figures 9A-9C show results obtained using primary AML blasts from three different patients with the HLA-A*02:01 allele. Figure 9D shows results obtained using a control sample (primary blasts lacking the specific HLA allele). [Figure 10] Figure 1 shows the editing frequencies obtained by simultaneously editing the TRAC and TRBC loci using multiple dual-guide RNAs. [Figure 11] Figure 11 shows the results of combinatorial editing using TRAC-targeted crRNA and TRBC-targeted crRNA. Figure 11A shows the efficiency of T cell receptor knockout. Figure 11B shows lentiviral transfection of the HD1-TCR construct into these cells. Figure 11C shows the expression of the TCR insert in edited T cells. Figure 11D shows the phenotype of TCR-edited T cells. [Figure 12] Figure 12 shows the editing frequency obtained by simultaneous editing of the TRAC locus and the TRBC locus using multiple single guide RNAs. Figure 12A shows the phenotypic rate of CD3 T cells after editing of the TRAC locus and the TRBC locus. Figure 11B shows the genotypic rate of indel formation at the TRAC locus and the TRBC locus of human CD3 T cells. [Figure 13]Figure 13A shows the extent of GFP insertion at the TRAC locus using a dual RNA guide and AAV vector (AV9) containing a gapped insertion template, as well as the extent of TRAC knockout using these dual RNA guides and AAV vectors that are CD3- (Figure 13B). Gapped insertion templates (or simply, gapped templates) contain two sequences flanked by a "gap," i.e., a region within the corresponding target sequence that is not present in the gapped insertion template. Gapped insertion templates are compatible with guides that target a site within the gap for cleavage, and are useful for quantitatively comparing such guides, regardless of whether they target the exact same cleavage site, so long as they both target a cleavage site within the gap. [Figure 14] Shown are the extent of GFP insertion at the TRAC locus using sgRNAs and gapped template AV9 (Figure 14A), and the percentage of cells engineered with these sgRNAs and gapped template AV9 that are phenotypically CD3- (Figure 14B). [Figure 15] Comparison of various promoter and inverted terminal repeat (ITR) lengths in TCR insertions as measured by positive tetramer staining (FIG. 15A) and mean fluorescence intensity (MFI) (FIG. 15B) during FACS analysis. [Figure 16] Figure 16 shows the amount of GFP expression driven from the endogenous TRAC promoter in cells engineered by the insertion protocol using AAV insertion of a promoterless GFP construct at the TRAC locus. Figure 16A shows a mock transfection control, Figure 16B shows RNP, and Figure 16C shows RNP + AV10-EGFP. X-axis: GFP expression; Y-axis: CD3e expression; measured by FACS. [Figure 17] 1 shows the insertion efficiency of TCR construct templates with and without promoter sequences (PL: promoterless; EF1a: promoter EF1 alpha or EF-1α) at ​​the TRAC locus. [Figure 18]Degranulation is measured in insert transformants using two different TCRs, with and without a promoter included in the transforming construct. [Figure 19] Interferon-γ expression is measured in insert transformants using two different TCRs, with and without a promoter included in the transforming construct. [Figure 20] Shown is the presence at the surface of two TCR constructs, one containing a Cys modification and one without. [Figure 21-1] Figures 21A-B show flow cytometry measurements of the degree of mismatching between the TCR chains of the inserted construct and the endogenous TCR chains. The FACS data in Figures 21A and 21B show the fraction of engineered cells expressing transgenic or mismatched TCRs, and the intensity of TCR expression in the engineered cells in Figures 21A and 21B is graphed as MFI in Figures 21C and 21D, respectively. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22] Flow cytometry is used to measure the degree of mismatching between the TCR chains of the inserted construct and the endogenous TCR chains in CD8+ or CD4+ cells. [Figure 23] VLD (Wilms tumor antigen) tetramer staining in four different TCR types with and without TRBC knockout is shown, demonstrating the extent of mispairing in WT1-TCR modified CD8+ cells. [Figure 24] VLD (Wilms tumor antigen) tetramer staining in four different TCRs with and without TRBC knockout is shown, demonstrating the extent of mispairing in WT1-TCR modified CD4+ cells. [Figure 25] Figure 1 shows the intensity of TCR expression in CD8+ cells when four TCR constructs were inserted into the TRAC locus, along with knockout of TRBCs, as measured by MFI of tetramer staining. [Figure 26] Figure 1 shows the intensity of TCR expression in CD4+ cells when four TCR constructs were inserted into the TRAC locus, along with knockout of TRBCs, as measured by MFI of tetramer staining. [Figure 27-1] Cytotoxicity of T cells containing inserted transgenic TCRs (AV11-TCR-A, AV13-TCR-B, AV12-TCR-C) and TRAC / TRBC double knockout or TRAC single knockout is shown. Cytotoxicity was measured by measuring fluorescence from apoptotic cells in response to caspase 3 / 7 apoptotic cell proliferation after 6 hours. [Figure 27-2] This is a continuation of Figure 27-1. [Figure 28-1] Cytotoxicity of T cells containing inserted transgenic TCRs (AV11-TCR-A, AV14-TCR-F, AV15-TCR-G, AV16-TCR-H) and TRAC / TRBC double knockout or TRAC single knockout was shown. Cytotoxicity was measured by measuring fluorescence from apoptotic cells in response to caspase 3 / 7 apoptotic cell proliferation after 6 hours. [Figure 28-2] This is a continuation of Figure 28-1. [Figure 29-1] Figure 1 shows the levels of peptide-specific IL-2 secretion from T cells containing inserted transgenic TCRs (AV11-TCR-A, AV13-TCR-B, AV12-TCR-C, AV14-TCR-F, AV15-TCR-G, AV16-TCR-H) and TRAC / TRBC double knockout or TRAC single knockout, as measured by ELISA. [Figure 29-2] This is a continuation of Figure 29-1. [Figure 29-3] This is a continuation of Figure 29-1. [Figure 29-4] This is a continuation of Figure 29-1. [Figure 30]Figure 1 shows the levels of peptide-specific IFN-γ secretion from T cells containing inserted transgenic TCRs (AV11-TCR-A, AV14-TCR-F, AV15-TCR-G, AV16-TCR-H) and TRAC / TRBC double knockout or TRAC single knockout, as measured by ELISA. [Figure 31] Figure 1 shows the levels of peptide-specific IFN-γ staining in CD4+ and CD8+ T cells containing inserted transgenic (AV11-TCR-A, AV14-TCR-F, AV15-TCR-G, AV16-TCR-H) TRAC / TRBC double knockout or TRAC single knockout. [Figure 32] 32A and 32B show the results of a CD107a degranulation assay, showing the degree of alloreactivity of CD4+ and CD8+ cells bearing transgenic TCRs (AV11-TCR-A, AV14-TCR-F, AV15-TCR-G, and AV16-TCR-H) at the TRAC locus, where native TRAC was knocked down, in the presence or absence of TRBC knockout. Figure 32A shows the percentage of CD107a+ staining cells among CD8+ T cells. Figure 31B shows the percentage of CD107a+ staining cells among CD4+ T cells. [Figure 33-1] Mixed lymphocyte reaction results are shown, showing the degree of alloreactivity of CD4+ and CD8+ cells carrying a transgenic TCR at the TRAC locus in which native TRAC was knocked down, in the presence or absence of TRBC knockout. CTV: CellTrace Violet (ThermoFisher). [Figure 33-2] This is a continuation of Figure 33-1. DETAILED DESCRIPTION OF THE INVENTION

[0126] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. Although the present teachings are described in connection with embodiments, it is not intended that the present teachings be limited to those embodiments. On the contrary, the present teachings are intended to be illustrative and not restrictive, and are not intended to be limiting unless otherwise specified. The present invention also encompasses forms and equivalent forms.

[0127] Before describing the present teachings in detail, it is important to note that the disclosure is not limited to particular compositions or process steps, and therefore It should be understood that the present invention may vary depending on the application. When used in scope, the singular forms "a," "an," and "the" are used interchangeably depending on the context. Unless explicitly stated otherwise, plural references are included. For example, when we say "a conjugate," we mean multiple conjugates, and A reference to a "cell" includes a plurality of cells, and so forth.

[0128] Numerical ranges are inclusive of the numbers defining the range, taking into account significant digits and errors inherent in measurements. Therefore, measured and measurable values ​​are understood to be approximate. "comprise", "comprises", "comprises" sing," "contain," "contains ", "containing," "include" , "includes," and "including The use of "" is not intended to be limiting. Both the above general description and the detailed description are illustrative and explanatory. It should be understood that this is for illustrative purposes only and is not intended as a limitation of the present teachings.

[0129] Unless otherwise specified herein, the terms "comprising" various components are used herein to refer to The embodiments in the present specification may be described as "consisting of" or "essentially consisting of" the listed components. and embodiments herein that state "consisting of" various components are also considered to be "composed of" various components. It is also considered to "comprise" or "consist essentially of" the listed ingredients; Embodiments herein that are described as "consisting essentially of" various components are intended to be illustrative and not restrictive of the components described. "comprising" or "consisting of" the recited components (this interchangeability is not guaranteed in the claims). (This does not apply to the use of these terms in this document.) Unless expressly indicated otherwise, the terms "and" "Or" is used herein in its inclusive sense, i.e., equivalent to "and / or." The term "about," when used before a list, modifies each member of that list. is doing.

[0130] The section headings used herein are for organizational purposes only and do not limit the scope of the intended subject matter. It should be understood that the materials incorporated by reference are not intended to limit the scope of the present specification. In the event of a conflict with any term defined in the document or other express content of this specification, this specification shall prevail. will be done.

[0131] I. Definition Unless otherwise stated, the following terms and expressions used herein have the following meanings: It is intended to.

[0132] "Polynucleotide" and "nucleic acid" as used herein refer to nucleic acids that are linked along a backbone. Nucleosides or nucleosides having linked nitrogen-containing heterocyclic bases or base analogues It refers to multimeric compounds containing analogues of RNA, DNA, mixed RNA-DNA, and The "backbone" of nucleic acids is made up of sugar-phosphodiester bonds, Peptide-nucleic acid conjugates ("peptide nucleic acids" or PNA; WO 95 / 32305) , phosphorothioate linkage, methylphosphonate linkage, or a combination thereof. The sugar moiety of nucleic acids can be ribose, deoxyribose, or or analogs containing substitutions (e.g., 2' methoxy or 2' halide substitutions) The nitrogenous bases can be any of the conventional bases (A, G, C, T, U), their analogs (e.g., For example, 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine modified uridine, etc.); inosine; purine or pyrimidine derivatives (e.g., N 4 -methyldeoxyguanosine, deazapurine or azapurine, deazapyrimidine or or azapyrimidine, pyrimidine bases with substituents at the 5- or 6-position (e.g., 5-methylpyrimidine, cytosine), purine bases with substituents at the 2-, 6-, or 8-positions, 2-amino-6 -methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine Imidine, 4-dimethylhydrazine-pyrimidine, and O 4 -Alkyl-pyrimidines; (U.S. Patent No. 5,378,825 and WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucle See "Chemical Acids" 5-36, Adams et al. (eds.), 11th ed., 1992. A nucleic acid is a single molecule if the backbone does not contain a nitrogenous base at each position in the polymer. or may contain multiple "abasic" residues (U.S. Patent No. 5,585,481). It may contain only traditional RNA or DNA sugars, bases, and linkages, or It may contain both conventional moieties and substitutions (e.g., a conventional moiety having a 2' methoxy substituent). A conventional nucleoside, or a conventional nucleoside and one or more nucleoside analogs (Polymers containing both.) Nucleic acids are locked into sugar conformations that mimic RNA. Analogues containing one or more LNA nucleotide monomers and a bicyclic furanose unit. These include "locked nucleic acids" (LNAs), which are nucleic acids that contain complementary RNA and DNA sequences. Enhances hybridization affinity to A sequences (Vester and Weng el, 2004, Biochemistry, Vol. 43(42): pp. 13233-41 ) RNA and DNA differ in their sugar moieties, and the presence of uracil or its analogues in RNA and the presence of thymine or its analogues in the DNA.

[0133] "Guide RNA," "gRNA," and simply "guide" as used herein Synonymously, crRNA (also known as CRISPR RNA), or crRNA and tRNA This refers to any combination of rRNA (also known as tracrRNA). The trRNA can be bound as a single RNA molecule (single-guide RNA, sgRNA), or may be combined as two separate RNA molecules (dual "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally occurring sequence or may be a sequence that is different from a naturally occurring sequence. The trRNA sequence may have modifications or alterations compared to the original trRNA sequence.

[0134] As used herein, a "guide sequence" refers to a sequence that is complementary to a target sequence and and guiding the binding or modification (e.g., cleavage) by the RNA-guided DNA binding agent. This refers to the sequence within the guide RNA that guides the RNA to the target sequence. " is sometimes referred to as a "targeting sequence" or a "spacer sequence." Streptococcus pyogenes (i.e., Spy Cas 9) and related Cas9 homologs / orthologs, the guide sequence is 20 base pairs. Shorter or longer sequences can also be used as guides, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length For example, in some embodiments, the guide sequence is selected from SEQ ID NOs: 1-178. At least 17, at least 18, at least 19, or at least 19 of the selected sequences In some embodiments, the target sequence comprises 20 consecutive nucleotides. or present on a chromosome or the like and is complementary to the guide sequence. The degree of complementarity or identity between the guide sequence and its corresponding target sequence is about 75%, about 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or For example, in some embodiments, the guide sequence may be At least 17, at least 18, at least 19 of the sequences selected from 1 to 178 or about 75%, about 80%, or about 85% for at least 20 consecutive nucleotides , about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% In some embodiments, the guide sequence and the target region comprise sequences that have 100 In other embodiments, the guide sequence and target region may be at least % complementary or identical. For example, the total length of the target sequence is at least 17, and at least If the length is at least 18, at least 19, at least 20, or more base pairs, The target sequence and the target sequence may contain 1, 2, 3, or 4 mismatches. In some embodiments, the guide sequences are at least 17, at least 18, or at least If the sequence contains at least 19, at least 20, or more nucleotides, it is considered a guide sequence. The target region may contain 1 to 4 mismatches. In some embodiments, If the guide sequence contains 20 nucleotides, the guide sequence and target region can be 1, 2, 3, or It may contain 1 or 4 mismatches.

[0135] Since the nucleic acid substrate for the RNA-guided DNA binder is a double-stranded nucleic acid, the RNA-guided The target sequences for the α-type DNA binders are the plus and minus strands of genomic DNA (i.e., That is, it includes both the given sequence and the reverse complement of that sequence. Therefore, when a guide sequence is said to be "complementary to a target sequence," the guide sequence is complementary to the target sequence. The guide RNA is induced to bind to the sense or antisense strand (e.g., reverse complement) of the target gene. It should be understood that in some embodiments, the guide sequence may be a target sequence. When the reverse complement of the guide sequence is bound to the reverse complement of the guide sequence, the T in the guide sequence is replaced with a U. The code sequence is identical to a specific group of nucleotides in the target sequence (e.g., does not include PAM).

[0136] As used herein, an "RNA-guided DNA binder" refers to a compound that binds RNA and DNA. A polypeptide or a complex of polypeptides having binding ability, or a complex of such a polypeptide The DNA-binding subunit is a sequence-specific subunit of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavage / nickel. The term "Cas DNA binder" refers to a Cas-nuclease and its inactive form ("dCas DNA binder"). "Creases," as used herein, include Cas creatinines, Cas nickases, and In some embodiments, the Cas cleavage agent or Cas nickases can cleave DNA via fusion with the FokI domain. The present invention also includes dCas DNA binders engineered to The Csm complex or Cm of the type III CRISPR system is used as a DNA binder for dCas and dCas. r complex, its Cas10 subunit, its Csm1 subunit, or its Cmr 2 subunits, the Cascade complex of type I CRISPR systems, its Cas3 subunit, and Class 2 Cas nucleases. 2Cas nuclease is a single-chain polypeptide with RNA-guided DNA binding ability. Class 2 Cas nucleases include RNA-guided DNA cleavage or Class 2 Cas cleavase / nickases that further possess nickases activity (e.g., H840 A variant, D10A variant, or N863A variant), and Class 2 dCas DNA binders with inactivated dCas / nickase activity Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, and C2 c2, C2c3, HF Cas9 protein (e.g., N497A variant, R661 A variant, Q695A variant, Q926A variant), HypaCas9 Protein (e.g., N692A variant, M694A variant, Q695A variant) , H698A variant), eSPCas9(1.0) protein (e.g., K810A variant, K1003A variant, R1060A variant), and eSPCas 9(1.1) protein (e.g., K848A variant, K1003A variant, R 1060A variant), and modified versions thereof. Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9. and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is See, e.g., Zetsche, Table S1 and See Table S3. See, e.g., Makarova et al., Nat Rev Microbiol ol, Vol. 13(11): pp. 722-36 (2015); Shmakov et al., Mole cular Cell, 60:385-397 (2015).

[0137] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" RNA-guided DNA binding agents such as Cas nucleases, e.g., Cas cleavase , Cas nickase, or dCas DNA binders (e.g., Cas9) In some embodiments, the guide RNA is an RNA such as Cas9. A guided DNA binding agent is guided to a target sequence, and the guide RNA hybridizes with the target sequence. and the binding agent binds to the target sequence; and the binding agent binds to the target sequence. If the bond is ze, cleavage or nicking can occur after binding.

[0138] As used herein, in a sequence comparison of a first sequence to a second sequence, the second sequence If X% or more of the positions in the primary sequence are shown to match, the primary sequence is considered to be a secondary sequence. A sequence is considered to "contain a sequence having at least X% identity to" a sequence. For example, The string AAGA contains sequences with 100% identity to the sequence AAG, which is A sequence comparison shows 100% identity because there is a match at all three positions in the two sequences. The difference between RNA and DNA (usually uridine vs thymidine) The presence of nucleoside analogues, such as uridines (or their replacements, or vice versa) or modified uridines, is As long as the nucleotides (such as thymidine, uridine, or modified uridine) have the same complement, (e.g., adenosine in all cases of thymidine, uridine, or modified uridine; Examples include cytosine and 5-methylcytosine, both of which are guanosine or modified guanosine as a complement), the difference in identity or complementarity between polynucleotides is Therefore, for example, when X is pseudouridine, N1-methylpseudouridine, or any modified uridine such as 5-methoxyuridine, 5'-AXG sequence, is perfectly complementary to the same sequence (5'-CAU), so 10 An exemplary alignment algorithm is Smith There are two algorithms: the S. Waterman algorithm and the Needleman-Wunsch algorithm. These are well known in the art. Therefore, it is within the skill of the art to determine whether alignment is appropriate for a given pair of sequences. generally of similar length and predicted amino acid identity of greater than 50% or For sequences with predicted nucleotide identity of more than 75%, see www.ebi.ac. The Needleman-Wunsch algorithm provided by EBI on the uk web server The Needleman-Wunsch algorithm, using the interface's initial settings, is usually adequate. It is sharp.

[0139] "mRNA," as used herein, refers to a gene that is translatable into a polypeptide (i.e., (aminoacyl-tRNA) that can serve as a substrate for translation by ribosomes and aminoacylated tRNAs mRNA refers to a polynucleotide containing an open reading frame. or analogs thereof (e.g., 2'-methoxyribose residues) In some embodiments, the sugars of the phosphate-sugar backbone of the mRNA are ribose residues, 2' -methoxyribose residues, or combinations thereof.

[0140] Exemplary guide sequences useful in the guide RNA compositions and methods described herein are: , Table 1, Table 2, and Table 3, and throughout this specification.

[0141] As used herein, an "indel" refers to a double-strand break (DSB) in a target nucleic acid. Refers to an insertion / deletion mutation, which consists of the insertion or deletion of several nucleotides at a position .

[0142] As used herein, "knockdown" refers to the knockdown of a particular gene product (e.g., a protein). Protein knockdown refers to a decrease in expression of a protein, mRNA, or both. The total cellular content of the protein from a target tissue or cell population mRNA knockdown can be measured by detecting the amount of mRNA. Methods for measuring the activity of mRNA isolated from a tissue or cell population of interest are known and include: Flow cytometry analysis is used to measure knockdown of protein expression. In some embodiments, "knockdown" refers to the process of knocking down a cell population. This may result in some reduction in the expression of a particular gene product, e.g., a reduction in the amount of transcribed mRNA or In some embodiments, "knockdown" may refer to a reduction in the amount of expressed protein. Specific gene products, such as the TRAC gene product or the TRBC gene product, on the cell surface It may refer to some decrease in the expression of a progenitor product.

[0143] As used herein, "knockout" refers to the deletion of a particular protein in a cell. Knockout refers to the reduction of expression of a protein in a cell, tissue, or population of cells. By detecting the total cellular amount, In some embodiments, the methods of the present invention involve determining whether a gene in one or more cells (e.g., For example, in a cell population), TRBC1, TRBC2, and / or TRAC are “knocked” In some embodiments, the knockout is a knockout of a T cell receptor in a cell. Expression of protein components (e.g., TRBC1, TRBC2, and / or TRAC) It is a complete loss, not the formation of a mutant T-cell receptor protein.

[0144] As used herein, "TRBC1" and "TRBC2" refer to T cell receptor Refers to two homologous genes encoding the β chains, with the T cell receptor β chain encoded by the TRBC1 gene or It is the gene product of the TRBC2 gene.

[0145] "TRBC" as used herein refers to TRBC1 and TRBC2.

[0146] The human wild-type TRBC1 sequence is available from NCBI Gene ID: 28639; Ensemb Available at l:ENSG00000211751. T-cell receptor Beta Constant, V_segment Translation Prod uct, BV05S1J2.2, TCRBC1, and TCRB are the genes of the TRBC1 gene. It is another name.

[0147] The human wild-type TRBC2 sequence is available from NCBI Gene ID: 28638; Ensemb Available at l:ENSG00000211772. T-cell receptor Beta Constant, V_segment Translation Prod uct, and TCRBC2 are alternative names for the TRBC2 gene.

[0148] The human wild-type TRAC sequence is available from NCBI Gene ID: 28755; Ensembl Available at: ENSG00000277734. T-cell receptor A lpha Constant, TCRA, IMD7, TRCA, and TRA are TRAC Another name for a gene.

[0149] Wilms tumor protein (sometimes called Wilms tumor antigen) is a protein that binds to the WT1 gene. The human wild-type WT1 sequence is available from NCBI Gene I D:7490;Ensembl:ENSG00000184937. GUD , AWT1, WAGR, WT33, NPHS4, and WIT-2 are synonyms for the WT1 gene. Wilms tumor protein is a protein that is found in solid tumors, including but not limited to Wilms tumor. It is expressed in various cancers, including hematological cancers (not found in the liver) and vascular endothelial cells of tumors. It has also been reported to be expressed in blastocysts and hematopoietic progenitor cells.

[0150] As used herein, a "target sequence" refers to a sequence complementary to the guide sequence of a gRNA. The interaction of the target sequence with the guide sequence results in: The RNA-guided DNA binder is directed to bind and nick or cleave within the target sequence. (depending on the activity of the binder)

[0151] As used herein, "treatment" refers to the treatment of a disease or disorder in a subject. refers to any administration or application of a drug to suppress or prevent the onset of the disease; alleviating one or more symptoms of the disease, curing the disease, or and preventing the recurrence of one or more symptoms of the disease.

[0152] The terms "about" or "approximately" refer to a particular value as determined by one of ordinary skill in the art. It refers to the allowable error in a measurement, depending on how the value is measured or determined. Partly depends.

[0153] II. Composition A. Compositions Comprising Guide RNA (gRNA) As used herein, for example, a guide RNA and an RNA-guided DNA binding agent (e.g., CR Using the ISPR / Cas system, in the TRBC gene and / or TRAC gene, Modify the DNA sequence (e.g., induce a single-strand break (SSB) or double-strand break (DSB) In some embodiments, the compositions are useful for treating TR. The AC sequence and / or TRBC sequence may be modified, and further, a TCR gene may be inserted. It is useful.

[0154] In some embodiments, the TRAC and / or TRBC sequences are modified and By inserting the TCR gene, the control (e.g., TRAC and / or TRBC) Favoured mispairing of TRAC and / or TRBC compared to unmodified controls For example, the TRBC sequence can be modified (e.g., by knocking out TRBCs), and By further inserting the TCR gene, mismatches between the inserted TCR and the native TRBCs can be prevented. Alternatively, the TRAC sequence may be modified (e.g., by knocking out TRAC). By inserting a TCR gene, the interplay between the inserted TCR and the native TRAC can be achieved. The guide sequences targeting the TRBC gene are shown in Table 1. The guide sequences targeting the TRAC gene are shown in SEQ ID NOs: 90 to 17 in Table 2. 8, 185, and 213-218.

[0155] In some embodiments, the genome is generated according to coordinates from the human reference genome hg38. The id sequences are complementary to the corresponding genomic regions shown in the table below. The guide sequence of the form is selected from the genome code listed in any of Tables 1, 2, and / or 3. For example, the guide sequences of further embodiments may be complementary to the sequences immediately adjacent to the guide sequences. The sequence may be one of the genomic coordinates listed in Table 1, Table 2, and / or Table 3. It may be complementary to a sequence comprising 5 contiguous nucleotides ±10 nucleotides.

[0156] SEQ ID NOs: 1 to 89, 90 to 178, 185, and 213 to 218 in Tables 1 and 2 Each guide sequence shown may contain additional nucleotides to form a crRNA. For example, the guide sequence may be followed at the 3' end by the following exemplary nucleotide sequence: Continued: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 400) (5'→3' In the case of sgRNA, the guide sequence may further contain additional nucleotides to A gRNA may be formed, for example, the following exemplary nucleotide sequence may be used as the guide sequence: followed at the 3' end by: GUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 401) (5' to 3' direction). The guide sequence contains an additional Further nucleotides may be included, for example, following the 3' end of the guide sequence: An sgRNA may be formed comprising the exemplary nucleotide sequence: GUUUUAGAGC UAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 402) (5'→3 'direction).

[0157] [Table 1] TIFF2025138662000002.tif173118 TIFF2025138662000003.tif163118

[0158] [Table 2] TIFF2025138662000005.tif174118TIFF2025138662000006.tif170118TIFF2025138662000007.tif12118

[0159] [Table 3] JPEG2025138662000009.jpg250161JPEG2025138662000010.jpg250161JPEG2025138662000011.jpg250161JPEG2025138662000012.jpg228170

[0160] In some embodiments, the present invention provides a method for the production of nucleases (e.g., Cas nucleases such as Cas9). The RNA-guided DNA binding agent can be a TRBC1, TRB 1 or 2, which contain a guide sequence that directs to a target DNA sequence within C2, and / or TRAC Compositions are provided that include multiple guide RNAs (gRNAs). The gRNAs may be any of those listed in Table 1 or Table 2. In some embodiments, the gRNA may comprise the following guide sequence: :(N) x GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG C (SEQ ID NO: 403), where "N" may be any natural or unnatural nucleotide. In addition, the entire N contains a TRBC guide sequence or a TRAC guide sequence as described herein. The gRNA may comprise a crRNA containing a guide sequence shown in Tables 1-3. The gRNAs consist of 17, 18, 19, or 2 of the guide sequences shown in Tables 1 and 2. In some embodiments, the gRNA may comprise a crRNA containing 0 consecutive nucleotides. A is a sequence of at least 17, at least 18, or at least 19 of the guide sequences shown in Table 1 and Table 2. About 75%, about 80%, or at least 19 or at least 20 consecutive nucleotides %, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or In some embodiments, the crRNA comprises a sequence having about 100% identity to the g The RNAs were approximately 75%, 80%, and 85% homologous to the guide sequences shown in Tables 1 and 2. , about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% The gRNA contains a crRNA that contains a sequence that has identity with the gRNA. In each embodiment described herein, the crRNA and trRNA may be a single They may bind as an RNA (sgRNA) or may be present on a separate RNA. In some cases, the sgRNA contains a crRNA component and a trRNA component. are covalently linked, for example, via a phosphodiester bond or other covalent bond. It's fine.

[0161] In each embodiment described herein, a "dual guide RNA" or "dgR A guide RNA may comprise two RNA molecules, e.g., a dgRNA. For example, a first RNA molecule comprising a crRNA containing a guide sequence shown in Table 1 and Table 2, and a t and a second RNA molecule comprising rRNA. The first RNA molecule and the second RNA molecule are covalently bonded. Although they may not be directly linked, base pairing between a portion of the crRNA and a portion of the trRNA The RNA may be double-stranded via the formation of a nucleotide sequence.

[0162] In each embodiment described herein, the guide RNA is referred to as a "single-guide RNA." " or "sgRNA." sgRNA may comprise one RNA molecule. A trRNA containing a guide sequence shown in Tables 1 and 2 covalently linked to the trRNA. The sgRNA may comprise a nucleotide sequence similar to that shown in Tables 1 and 2. Contains 17, 18, 19, or 20 consecutive nucleotides of the guide sequence In some embodiments, the crRNA and the trRNA are shared via a linker. In some embodiments, the sgRNA is covalently linked to a portion of the crRNA. It forms a stem-loop structure through base pairing with a part of the trRNA. In some embodiments, the crRNA and the trRNA are linked by one or more non-phosphodiester bonds. are covalently linked via multiple bonds.

[0163] In some embodiments, the trRNA is a trRNA derived from a naturally occurring CRISPR / Cas system. In some embodiments, the trRNA may comprise all or part of a trRNA sequence. contains truncated or modified wild-type trRNA. The length of the trRNA depends on the CR used. In some embodiments, the trRNA is 5, 6, 7, or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides In some embodiments, the trRNA comprises, or consists of, e.g., one or Multiple hairpin or stem-loop structures, or one or more bulge structures, etc. It may also contain certain secondary structures.

[0164] In some embodiments, the present invention provides the nucleic acids of SEQ ID NOs: 1-89 and SEQ ID NOs: 90-178. Compositions comprising one or more guide RNAs comprising any one of the guide sequences are provided.

[0165] In some embodiments, the present invention provides a method for the preparation of a medicament comprising any one of SEQ ID NOs: 179-184. Alternatively, a composition comprising multiple sgRNAs is provided.

[0166] In one embodiment, the present invention provides SEQ ID NOs: 1-89, 90-178, 185, and 213. At least 99%, at least 98%, at least 9% of any of ~218 nucleic acids 7%, at least 96%, at least 95%, at least 94%, at least 93%, a guide sequence having at least 92%, at least 91%, or at least 90% identity to the A composition comprising a gRNA comprising a sequence is provided.

[0167] In other embodiments, the composition comprises SEQ ID NOs: 1-89, 90-178, 185, and Contains guide sequences selected from any two or more of the guide sequences of 213 to 218 In some embodiments, the gene comprises at least one (e.g., at least two) gRNAs. The compositions include nucleic acids of SEQ ID NOs: 1-89, 90-178, 185, and 213-218. At least 99%, at least 98%, at least 97%, or at least At least 96%, at least 95%, at least 94%, at least 93%, at least 92% , at least 91%, or at least 90% identity to the guide sequence, respectively. It contains at least two gRNAs.

[0168] The guide RNA composition of the present invention is a composition that encodes the TRBC1 gene, the TRBC2 gene, and / or is designed to recognize (e.g., hybridize to) a target sequence within the TRAC gene. For example, a TRBC1 target sequence, a TRBC2 target sequence, and / or a TRAIL target sequence may be designed. The target sequence is recognized and cleaved by the provided Cas cleavage base containing the guide RNA. In some embodiments, RNA-guided DNA fragments, such as Cas fragments, can be used. The combination drug is a guide RNA that targets the TRBC1 gene, the TRBC2 gene, and / or The guide sequence of the guide RNA may be directed to the target sequence of the TRAC gene. is hybridized to the target sequence, and an RNA-guided DNA such as the above-mentioned Cas cleavage base is generated. The A binder cleaves the target sequence.

[0169] In some embodiments, the selection of one or more guide RNAs is based on the TRBC1 gene, The target sequence is determined based on the target sequence within the TRBC2 gene and / or the TRAC gene.

[0170] Without being bound by any particular theory, it is believed that the Mutations (e.g., fragments caused by indels resulting from nuclease-mediated DSBs) Although some mutations (e.g., rham-shift mutations) may be less tolerated than mutations in other regions of the gene, Therefore, the location of the DSB may influence the amount or type of protein knockdown that can be caused. In some embodiments, TRBC1, TRBC2, and and / or using gRNAs that are complementary to or have complementarity with target sequences within TRAC. and R at specific sites in the appropriate TRBC1, TRBC2, and / or TRAC genes. In some embodiments, the gRNA is a TRB-guided DNA binder. C1 and / or TRBC2 exon 1, 2, 3, or The target sequence in the fourth exon and / or the first and second exons of TRAC complementary to a target sequence in the exon, the third exon, or the fourth exon; Alternatively, the nucleic acid sequence may be designed to have a guide sequence complementary to the nucleic acid sequence.

[0171] In some embodiments, the guide sequence is selected from the group consisting of the human TRBC1 gene, the human TRBC2 gene, gene, and / or at least 99% against target sequences present in the human TRAC gene %, at least 98%, at least 97%, at least 96%, at least 95%, at least at least 94%, at least 93%, at least 92%, at least 91%, or less In some embodiments, the target sequence is the same as the guide RNA. In some embodiments, the guide sequence of the guide RNA may be complementary to the guide sequence of the guide RNA. The degree of complementarity or identity between a code sequence and its corresponding target sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least It may be 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the target sequence and the guide sequence of the gRNA are 100% complementary or In other embodiments, the target sequence and the guide sequence of the gRNA may be at least For example, if the total length of the guide sequence is 20, The target sequence and the guide sequence of the gRNA contain one, two, three, or four mismatches. In some embodiments, the target sequence and the guide sequence of the gRNA may be If the string is 20 nucleotides, it may contain 1 to 4 mismatches.

[0172] In some embodiments, the compositions or formulations disclosed herein comprise Open reading frames encoding RNA-guided DNA binders such as Cas nucleases In some embodiments, the Cas gene includes an mRNA containing an open reading frame (ORF). mRNA containing an ORF encoding an RNA-guided DNA binder such as a nuclease is provided. are prepared, used, and administered.

[0173] B. Modified gRNA and mRNA In some embodiments, the gRNA is chemically modified. The gRNA may contain one or more modified nucleosides or nucleotides, and may be referred to as "modified" These are called gRNAs or "chemically modified" gRNAs, and contain nucleotides that replace the standard A, G, C, and U residues. Instead of or in addition to one or more non-naturally occurring and / or may refer to the presence of naturally occurring components or configurations. In this case, modified gRNAs are synthesized using non-standard nucleosides or nucleotides, Modified nucleosides and nucleotides are referred to herein as "modified." (i) phosphodiester backbone linkages, resulting in linkages One or both of the free phosphates and / or one or both of the bound phosphates (ii) changes in the ribose sugar, e.g., substitutions (exemplary backbone modifications); For example, alterations, e.g., substitutions, of the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); ) extensive replacement of the phosphate moiety with a “dephospho” linker (an exemplary backbone modification); (iv) Modifications or substitutions of naturally occurring nucleobases, including those with non-standard nucleobases (exemplary bases are (v) ribose-phosphate backbone substitutions or modifications (exemplary backbone modifications); (vi) Modification of the 3' or 5' end of an oligonucleotide, e.g., removal of the terminal phosphate group, modification Or a modification, or substitution, or a moiety, cap, or linker connection (such as 3' or or 5' cap modifications may include sugar modifications and / or backbone modifications; and (vii ) Sugar modifications or substitutions (exemplary sugar modifications).

[0174] Chemical modifications such as those listed above can be combined to form two, three, four or more Modifications, including nucleosides and nucleotides (collectively "residues"), which may have the above modifications. For example, modified residues can be modified sugars. and modified nucleobases. In some embodiments, all bases of the gRNA are modified. For example, all bases have modified phosphate groups, such as phosphorothioate groups. In certain embodiments, all, or substantially all, of the phosphate groups of the gRNA molecule are phosphodiesterases. In some embodiments, the modified gRNA is an RNA In some embodiments, the nucleotide sequence comprises at least one modified residue at or near the 5' end of the nucleotide sequence. , the modified gRNA contains at least one modified residue at or near the 3' end of the RNA. .

[0175] In some embodiments, the gRNA comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least at least 5%, at least 10%, at least 15%, at least 20%, at least 25 %, at least 30%, at least 35%, at least 40%, at least 45%, at least at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) are modified nucleosides or nucleotides .

[0176] Unmodified nucleic acids can be degraded by, for example, intracellular nucleases or nucleases present in serum. For example, nucleases may be able to degrade nucleic acids by phosphodiesterases. In accordance with this, in one embodiment, the g RNA is stabilized against intracellular or serum-based nucleases It may contain one or more modified nucleosides or nucleotides, for example to In some embodiments, the modified gRNA molecules described herein are useful in in vivo and in vivo experiments. They may exhibit a reduced innate immune response when introduced into cell populations both ex vivo and in vivo. "Innate immune response" encompasses cellular responses to foreign nucleic acids, including single-stranded nucleic acids, The response involves the expression and release of cytokines (especially interferons) and the induction of cell death. include.

[0177] In some embodiments of the backbone modification, the phosphate group of the modified residue is substituted with one or more of its oxygens. Further, modified residues (e.g., modified nuclei) can be modified by replacing the The modified residue present in the unmodified phosphate moiety is a modified phosphate moiety as described herein. In some embodiments, the backbone modification of the phosphate backbone may include extensive substitution with acid groups. may include changes that result in uncharged linkers or charged linkers with asymmetric charge distribution .

[0178] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, and boranophosphates. esters, boranophosphate esters, hydrogen phosphonates, phosphoramidates , alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom in the phosphate group is achiral. Substitution of one of the bridging oxygens with one of the atoms or groups of atoms listed above gives the phosphorous acid Asymmetric phosphorous atoms can be chiral. The molecule may be in the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). The backbone may have bridging oxygens (i.e., oxygens connecting the phosphates to the nucleosides). oxygen), nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) ), and carbon (bridging methylene phosphonate). can occur on either oxygen at the linkage, or It can also occur with both oxygens present.

[0179] The phosphate group can be replaced with a non-phosphorus-containing linker in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced with a neutral moiety. Examples of moieties include methylphosphonic acid, hydroxylamino, siloxane, carbonate, Carboxymethyl, carbamic acid, amide, thioether, ethylene oxide linker, Sulfonic acids, sulfonamides, thioformacetals, formacetals, oximes, methyl ethyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0180] Nuclease-resistant alternative nucleosides or alternative nucleoside linkers and ribose sugars It is also possible to construct scaffolds that mimic nucleic acids, substituted with nucleotides. In some embodiments, the nucleobases are linked to alternative backbones. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (P Alternative nucleosides include, but are not limited to, nucleosides such as NA.

[0181] Modified nucleosides and nucleotides may contain one or more modifications to the sugar group. For example, the 2' hydroxyl group (OH) can be modified can be substituted with, for example, several different "oxy" or "deoxy" substituents. In some embodiments, the modification to the 2' hydroxyl group can be Because silyl cannot form a 2'-alkoxide ion by deprotonation , the stability of the nucleic acid can be enhanced.

[0182] Examples of 2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl. , or sugar); polyethylene glycol (PEG), O(CHCH 2O) n CH2CH2OR (wherein R is, for example, H or an optionally substituted alkyl group) n can be a number between 0 and 20 (e.g., 0-4, 0-8, 0-10, 0-16, 1 ~4, 1~8, 1~10, 1~16, 1~20, 2~4, 2~8, 2~10, 2~16, can be an integer between 2 and 20, 4 and 8, 4 and 10, 4 and 16, and 4 and 20). In some embodiments, the 2' hydroxyl group modification can include 2'-O In some embodiments, the 2' hydroxyl group modification can be 2' -Me. The hydroxyl group can be replaced with fluoride, resulting in a 2'-fluoro modification. In some embodiments, the 2' hydroxyl group modification may comprise a "locked" nucleic acid (LNA). The 2' hydroxyl can be bridged with a C alkylene bridge or C 1-6 Heteroalkyl The ribose sugars can be linked to the 4' carbon of the same ribose sugar by a ribosomal bridge or the like, and exemplary bridges are Examples include methylene bridges, propylene bridges, ether bridges, or amino bridges. 2' hydroxyl group modification can be performed to O-amino (amino is, for example, NH2; alkylamino, dialkylamino, heterocyclyl, aryl amino, diarylamino, heteroarylamino, or diheteroarylamino, ethylene ethylenediamine, or polyamino), and aminoalkoxy, O(CH2) n -amino (amino is, for example, NH2; alkylamino, dialkylamino) heterocyclic group, arylamino, diarylamino, heteroarylamino, or diheterocyclic group arylamino, ethylenediamine, or polyamino) In some embodiments, the 2' hydroxyl group modification can be performed at C2 of the ribose ring. It can include "unlocked" nucleic acids (UNAs) that lack the -C3 bond. In some embodiments, the 2' hydroxyl group modification is a methoxyethyl group (MOE) (OC H2CH2OCH3, e.g., PEG derivatives).

[0183] The "deoxy" 2' modification is a modification of the 2' amino acid sequence by hydrogen (i.e., in the overhanging portion of dsRNA, etc.). deoxyribose sugars; halo (e.g., bromo, chloro, fluoro, or iodo) ); amino (amino is, for example, NH2; alkylamino, dialkylamino, heterocyclic group , arylamino, diarylamino, heteroarylamino, diheteroarylamino , or an amino acid); NH(CH2CH2NH) n CH2CH2-A amino (amino can be, for example, as described herein), —NHC(O) R (R is, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl) or sugar), cyano; mercapto; alkyl-thio-alkyl; thi and optionally substituted with amino, etc., as described herein. alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which may be It can include.

[0184] Sugar modifications are carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. That is, modified nucleic acids can include sugar groups that may contain one or more of the following: These modified nucleic acids may contain nucleotides containing arabinose or the like as the nucleotide. These abasic sugars may also contain abasic sugars at one or more of the constituent sugar atoms. Modified nucleic acids can also contain one or more sugars that are in the L-form. can be used (e.g., L-nucleosides).

[0185] The modified nucleosides and modified nucleosides described herein that can be incorporated into modified nucleic acids are The nucleic acids may contain modified bases (also known as nucleobases). Examples of nucleobases include: These include adenine (A), guanine (G), cytosine (C), and uracil (U) However, the present invention is not limited to these. By modifying or completely substituting these nucleic acid bases, Modified residues can be obtained that can be incorporated into modified nucleic acids. are independently selected from a purine, a pyrimidine, a purine analog, or a pyrimidine analog. In some embodiments, the nucleobases may be, for example, naturally occurring derivatives and synthetic bases. It may contain synthetic derivatives.

[0186] In embodiments using dual guide RNAs, crRNA and tracr RNA Each of the sequences may contain modifications. Such modifications may be incorporated into the crRNA and / or or at one or both ends of the tracrRNA. In this embodiment, one or more residues at one or both ends of the sgRNA are chemically modified. and / or the internal nucleosides may be modified, and / or The entire sgRNA may be chemically modified. Certain embodiments include 5' end modifications. Certain embodiments include a 3' end modification. Certain embodiments include a 5' end modification and Contains 3' end modifications.

[0187] In some embodiments, the guide RNA disclosed herein is The term "Chemically Modified International Publication No. 2, filed December 8, 2017, entitled "Guide RNAs" Some embodiments include one of the modification patterns disclosed in US Pat. No. 5,699,028. The guide RNAs disclosed herein are incorporated by reference in their entirety. The structure / structure disclosed in U.S. Patent Application Publication No. 20170114334, which is incorporated herein by reference. In some embodiments, the modified amino acid sequence comprises one of the modified amino acid sequences disclosed herein. The RNA is disclosed in International Publication No. 2017, the entire contents of which are incorporated herein by reference. It includes one of the structure / modification patterns disclosed in US Pat. No. 6,236,794.

[0188] In some embodiments, the sgRNA has any of the modification patterns presented herein. N is any natural or unnatural nucleotide, and N collectively represents any of the nucleotides in Tables 1-3 of the present specification. TRBC1 guide sequence, TRBC2 guide sequence, and / or T In some embodiments, the modified sgRNA comprises the following sequence: Contains: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGm CmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGU CCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU(Sequence number No. 300), where "N" can be any natural or unnatural nucleotide, and N as a whole , TRBC1 as described in Tables 1 and 2, or as described herein Construct a guide sequence, a TRBC2 guide sequence, and / or a TRAC guide sequence. For example, SEQ ID NO: 300 is included herein, and N is a sequence number listed in Tables 1 and 2 herein. The guide sequences are replaced with any of the sequences shown (SEQ ID NOs: 1-89, 90-178). , 185, and 213–218).

[0189] Any of the following modifications may be present in the gRNAs and mRNAs described herein: Good too.

[0190] The terms "mA," "mC," "mU," or "mG" refer to 2'-O-Me modified It can be used to represent a nucleotide.

[0191] The 2'-O-methyl modification can be shown as follows: [ka]

[0192] Another chemical modification that has been shown to affect the nucleotide sugar ring is the halogen substitution. For example, 2'-fluoro (2'-F) substitutions on the nucleotide sugar ring can be used to Nucleotide binding affinity and nuclease stability can be increased.

[0193] As used herein, the terms "fA," "fC," "fU," or "fG" refer to 2'-F substituted amino acids. can be used to refer to the substituted nucleotide.

[0194] The 2'-F substitution can be shown as follows: [ka]

[0195] Phosphorothioate (PS) bonds are bonds between phospho- and nucleotide bases. One oxygen atom that is not bridging the phosphate in the diester bond is replaced with sulfur. When phosphorothioates are used to make oligonucleotides, The modified oligonucleotides are sometimes referred to as S-oligos.

[0196] " * " may be used to refer to PS modification. In this application, the term A * , C * , U * , or G * represents a nucleotide linked to the next (e.g., 3') nucleotide by a PS bond. It can be used to represent.

[0197] In this application, the term "mA * "," mC * ", "mU * " or "mG * " is 2' -O-Me and linked to the next (e.g., 3') nucleotide by a PS bond. It can also be used to represent a nucleotide.

[0198] The diagram below shows the phosphodiester bond formed by S-substitution of the non-bridging phosphate oxygen. This shows the creation of PS bonds instead of [ka]

[0199] An abasic nucleotide is a nucleotide that lacks a nitrogenous base. It represents an oligonucleotide with a base site (also known as an apurinic site). [ka]

[0200] An inverted base is a base that is reversed from the normal 5'→3' bond. A base having a 5'→5' or 3'→3' bond. For example, The following is the result. [ka]

[0201] The abasic nucleotide can be attached in a reverse bond. The nucleotide may be attached to the 5'-terminal nucleotide via a 5'→5' linkage, or Alternatively, an abasic nucleotide may be attached to the 3'-terminal nucleotide via a 3'→3' linkage. Inverted abasic nucleoside at the 5' or 3' terminal nucleotide The tide is sometimes referred to as an inverted abasic end cap.

[0202] In some embodiments, the first 3, 4, or 5 nucleotides of the 5' end one or more of the last three, four, or five nucleotides at the 3' end In some embodiments, the modifications are for stability and / or activity. 2'-O-Me, 2'-F, inverted abasic nucleotides, PS bonds for increased activity or other nucleotide modifications well known in the art.

[0203] In some embodiments, the first four nucleotides of the 5' end and the last nucleotide of the 3' end The four nucleotides are linked by phosphorothioate (PS) bonds.

[0204] In some embodiments, the first three nucleotides at the 5' end and the last nucleotide at the 3' end Three of the nucleotides include 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end Some nucleotides include 2'-fluoro (2'-F) modified nucleotides. In an embodiment, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end The nucleotide comprises an inverted abasic nucleotide.

[0205] In some embodiments, the guide RNA comprises a modified sgRNA. wherein the sgRNA comprises the modification pattern set forth in SEQ ID NO: 401, where N is any N is a natural or non-natural nucleotide, and N is a nucleotide selected from the group consisting of nucleotides such as those shown in Tables 1 and 2. Thus, the nuclease is directed to a target sequence within TRBC1, TRBC2, and / or TRAC. A guide sequence is constructed to induce the gene.

[0206] In some embodiments, the guide RNA is set forth in any one of SEQ ID NOs: 179-184. In some embodiments, the guide RNA comprises an sgRNA selected from the group consisting of SEQ ID NOs: 1-17. an sgRNA comprising any one of the eight guide sequences and the nucleotide sequence of SEQ ID NO: 401; The nucleotide of SEQ ID NO: 401 is located at the 3' end of the target guide sequence. wherein the sgRNA is modified as shown herein or in SEQ ID NO: 300. Good too.

[0207] As mentioned above, in some embodiments, the compositions or The formulations may comprise an RNA-guided DNA-binding agent, such as a Cas nuclease, as described herein. It contains an mRNA containing an open reading frame (ORF) that encodes the In some forms, ORs encode RNA-guided DNA-binding agents such as Cas nucleases. In some embodiments, an mRNA comprising F is provided, used, or administered. The ORF encoding the guided DNA nuclease is called a "modified RNA-guided DNA binder ORF,” or simply used as shorthand to indicate that the ORF is modified It is a "modified ORF" that is

[0208] In some embodiments, the modified ORFs are at least one, more than one, or all of the uridi In some embodiments, the modified uridine may comprise a modified uridine at the 5-position. In some cases, it is a uridine modified with halogen, methyl, or ethyl. In embodiments, the modified uridine is modified at the 1-position with, for example, a halogen, methyl, or ethyl. The modified uridine is pseudouridine, N1-methyl- Pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof In some embodiments, the modified uridine is a 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. The modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridines are pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is N1-methylpseudo In some embodiments, the modified uridine is a combination of uridine and 5-methoxyuridine. Uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine In some embodiments, the modified uridine is a mixture of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5- In combination with methoxyuridine.

[0209] In some embodiments, the mRNA disclosed herein is Cap 0, Cap 1 , or cap 2. The 5' cap is usually located at the 5' end of the mRNA. → 7-methylglucan bound to the 5' position of the first nucleotide of the 3' strand via 5'-triphosphate ribonucleotides (e.g., further modified as described below for ARCA) (It may be the first cap-proximal nucleotide). Both the ribose of the first cap-proximal nucleotide and the ribose of the second cap-proximal nucleotide Cap 1 contains a 2'-hydroxyl group. The ribose of the two transcribed nucleotides has a 2'-methoxy and a 2'-hydroxyl group, respectively. Cap 2 contains the first cap-proximal nucleotide and the second cap-proximal nucleotide of the mRNA. Both riboses of the paranucleotides contain 2'-methoxy. 2014), Proc Natl Acad Sci USA, Vol. 111(No. 33): pp. 12025-30; Abbas et al. (2017) Proc Natl Acad Sc See i USA, vol. 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as A, contain cap 1. or Cap 2. Cap 0 and other caps other than Cap 1 and Cap 2 The cap structure is "protected" by components of the innate immune system, such as IFIT-1 and IFIT-5. This results in increased cytokine levels, including type I interferons. IFIT-1 and IFIT-2 may be immunogenic in mammals, such as humans, because they may be elevated in their activity. Components of the innate immune system, such as IFIT-5 and IFIT-6, encode either cap 1 or cap 2 of mRNA. It may also compete with eIF4E for binding to caps other than the mR NA translation may be inhibited.

[0210] Caps can be included at the same time as transfer. For example, ARCA (Anti-Reverse rse Cap Analog; Thermo Fisher Scientific Catalog The 5'-position of the guanine ribonucleotide is linked to 7-methylguanine. Cap analog containing 3'-methoxy-5'-triphosphate of thiamin, ARCA can be incorporated into transcripts at the initiation stage. This results in a cap 0 cap where the 2' position of the thiol is a hydroxyl. Pinski et al. (2001), "Synthesis and propertie s of mRNAs containing the novel 'anti-re verse' cap analogs 7-methyl(3'-O-methyl) GpppG and 7-methyl(3'deoxy)GpppG,” RNA, Vol. 7 See pages 1486-1495. The ARCA structure is shown below. [ka]

[0211] CleanCap(TM)AG(m7G(5')ppp(5')(2'OMeA)pG TriLink Biotechnologies ies) Catalog Number N-7113) or CleanCap™ GG (m7G (5 ')ppp(5')(2'OMeG)pG; TriLink Biotechnologies, Inc. By using the log number N-7133, the cap 1 structure can be obtained simultaneously with transcription. The 3'-O-methyl group of CleanCap™ AG and CleanCap™ GG Chilled versions are also available from TriLink Biotechnologies, Inc., catalog number N-7 Available as N-413 and N-7433. CleanCap™ AG structure is shown below. Shown below. [ka]

[0212] Alternatively, a cap can be added to the RNA after transcription. Capping enzyme is commercially available (New England Biolabs, Inc.). England Biolabs (catalog number M2080S), and its D1 subunit RNA triphosphatase and guanylyltransferase activities conferred by and the guanine methyltransferase conferred by its D12 subunit. Therefore, the enzyme converts S-adenosylmethylcellulose to cap 0. In the presence of thionine and GTP, 7-methylguanine can be added to RNA See, for example, Guo, P. and Moss, B. (1990), Proc. Natl. A cad.Sci.USA, vol. 87, pp. 4023-4027;Mao, X. and Shum an, S. (1994), J. Biol. Chem., vol. 269, pp. 24472-2447 See 479.

[0213] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the poly-A tail comprises at least 20, at least 30 pcs, at least 40 pcs, at least 50 pcs, at least 60 pcs, at least 70 pcs, at least 80, at least 90, or at least 100 adenines, optionally 3 In some embodiments, the poly-A tail contains up to 95,000 adenines. It contains 96, 97, 98, 99, or 100 adenine nucleotides.

[0214] C. Ribonucleoprotein complex In some embodiments, a 1000 mg / kg bwt. ... One or more gRNAs, or one or more sgRNAs from Table 3, and an RNA guide and nucleases, such as Cas nucleases, such as Cas9. In some embodiments, the RNA-guided DNA binding agent (e.g., Cas9) has a double-stranded endonuclease activity, sometimes referred to as a cleave endonuclease activity. In some embodiments, the RNA-guided DNA binding agent has Cas9 nuclease activity. Examples of Cas9 nucleases include those from Streptococcus pyogenes (S. pyogenes). enes, Staphylococcus aureus (S. aureus), and other prokaryotes (e.g., Cas9 nucleases of type II CRISPR systems (see the list in the paragraph above) and modifications thereof (e.g., engineered or mutated) forms. See, for example, U.S. Patent Application Publication No. 2016 / 004999. See US Pat. Nos. 312198 and 2016 / 0312199. Cas nucleases Other examples include the Csm complex or Cmr complex of a type III CRISPR system, is the Cas10 subunit, Csm1 subunit, or Cmr2 subunit and the Cascade complex of the type I CRISPR system, or its Cas3 subunit, In some embodiments, the Cas nuclease is a type IIA, type IIB, or Considerations for various CRISPR systems and Cas nucleases For information on this, see, for example, Makarova et al., Nat. Rev. Microbiol. 9: pp. 467-477 (2011); Makarova et al., Nat. Rev. Mic robiol, vol. 13: pp. 722-36 (2015); Shmakov et al., Molec See ular Cell, 60:385-397 (2015).

[0215] Non-limiting exemplary species from which Cas nucleases can be obtained include: Streptococcus pyogenes, Strep Streptococcus thermophilus ), Streptococcus sp., Staphylococcus aureus Staphylococcus aureus, Listeria innocua teria innocua), Lactobacillus gasseri (Lactobacillus gasseri), Francisella novici da), Wolinella succinogenes ), Sutterella wadsworthensi s), Gammaproteobacterium, meninges Neisseria meningitidis, Campylobacter jeju Campylobacter jejuni, Pasteurella multocida eurella multocida), Fibrobacter succinogenes (Fibr obacter succinogene), Rhodospirillum rubrum (Rhodos pirillum rubrum), Nocardiopsis dassonvillei (Nocardi opsis dassonvillei), Streptomyces pristinaeaspira Squirrel (Streptomyces pristinaespiralis), Strep Streptomyces viridochromogenes genes), Streptomyces viridochromogenes (Streptomyces viridochromogenes), Streptosporangium roseum (Str Streptosporangium roseum) (Streptosporangium roseum), Alicyclobacillus acidi Caldarius (Alicyclobacillus acidocaldarius), Bacillus pseudomycoides , Bacillus selenitireduce ns), Exiguobacterium sibiricum (Exiguobacterium si biricum, Lactobacillus delbruec kii), Lactobacillus salivaryus arius), Lactobacillus buchneri ri), Treponema denticola, Mik Microscilla marina, a Burkholderiales bacterium ( Burkholderiales bacterium), Polaromonas naphthalenivo Lance (Polaromonas naphthalenivorans), Polaromona Polaromonas sp., Crocosphaera watsonii (Croc osphaera watsonii), Cyanothece species sp.), Microcystis aeruginosa (Microcystis aeruginosa sa), Synechococcus sp., Acetohalobi Acetohalobium arabaticum, Ammonium Ammonifex degensii, Carbicella ulcerata Tar becsci (Caldicelulosiruptor becscii), Candida Candidatus Desulforudis, Botulism Clostridium botulinum, Clostridium difficile Clostridium difficile, Finegoldia magna (F inegoldia magna), Natranaerobius thermophilus (Natra naerobius thermophilus), Pelotomaculum thermopropio Pelotomaculum thermopropionicum, Assisi Acidithiobacillus caldus, reed Dithiobacillus ferrooxidans (Acidithiobacillus ferr ooxidans), Allochromatium vinosum (Allochromatium v inosum), Marinobacter sp., Nitro Nitrosococcus halophilus, Nitrosococcus Nitrosococcus watsoni, pseudomonad Alteromonas haloplanktis (Pseudoalteromonas halop lanktis), Ktedonobacter racemifur (Ktedonobacter r acemifer), Methanohalobium ebestigatum (Methanohalobi um evestigatum), Anabaena variabilis (Anabaena var iabilis, Nodularia spumigen a), Nostoc sp., Arthrospira maxima hrospira maxima), Arthrospira platensis (Arthrosp ira platensis), Arthrospira sp. .), Lyngbya sp., Microcoleus kutnoplastacea Microcoleus chthonoplastes, Oscillatoria species ( Oscillatoria sp.), Petrotoga mobilis (Petrotoga m obilis), Thermosipho africa nus), Streptococcus pasteurianus (Streptococcus pa steurianus), Neisseria cinerea ), Campylobacter lari, Parvivac Parvibaculum lavamentivoran s), Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae ( Lachnospiraceae) bacterium ND2006, and Acaryochloris marina (Acaryochloris marina).

[0216] In some embodiments, the Cas nuclease is selected from the group consisting of Streptococcus pyogenes (Streptococcus pyogenes) and Streptococcus pyogenes. Cas9 nuclease from Bacillus pyogenes. In the cytoplasmic state, Cas nucleases are expressed in Streptococcus thermophilus (Streptococcus The Cas9 nuclease is derived from Coccus thermophilus. In some embodiments, the Cas nuclease is a nuclease specific for Neisseria meningitidis. In some embodiments, the Cas9 nuclease is derived from Cas9. The nuclease is derived from Staphylococcus aureus In some embodiments, the Cas nuclease is a Cas9 nuclease of Fragment. Cpf1 nucleic acid from Francisella novicida In some embodiments, the Cas nuclease is an Acidaminococcus nuclease. Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is selected from the group consisting of Lachnospiraceae (Lachnosp In another embodiment, the Cpf1 nuclease is derived from the bacterium Cpf1 (Iraceae). Cas nuclease is a nuclease that binds to the tularemia bacterium Francisella tularensis ), Lachnospiraceae bacteria, Butyrivibrio protein Butyrivibrio proteoclasticus, Pele Peregrinibacteria bacteria, Parcubacteria (Parcubacteria) bacteria, Smithella, Acidamino Acidaminococcus, Candidatus Methanoplasma ter Mitsumu (Candidatus Methanoplasma termitum), - Eubacterium eligens, Moraxella · Moraxella bovoculi, Leptospira · Leptospira ptospira inadai, Porphyromonas cleviolicanis (Porph yromonas crevioricanis), Prevotella dysciens (Pre votella disiens, or Porphyromonas macacae (Porphy Cpf1 nuclease from C. romonas macacae. In terms of morphology, Cas nucleases are found in the genus Acidaminococcus. us) or Cpf1 nucleic acids from the Lachnospiraceae family It's Aze.

[0217] In some embodiments, the gRNA and the RNA-guided DNA-binding agent are combined. In some embodiments, RNA is a protein that is called a ribonucleoprotein complex (RNP). The guided DNA binding agent is a Cas nuclease. The combination of a Cas nuclease and a Cas RNP is called a Cas RNP. In embodiments, the RNP comprises type I, type II, or type III components. In its morphology, Cas nucleases are the precursors of the Cas9 protein from type II CRISPR / Cas systems. In some embodiments, the gRNA and Cas9 together are Ca It is called s9 RNP.

[0218] Wild-type Cas9 has two nuclease domains (RuvC and HNH). The uvC domain cleaves non-target DNA strands, and the HNH domain cleaves target DNA strands. In some embodiments, the Cas9 protein comprises two or more RuvC domains and / or or two or more HNH domains. In some embodiments, the Cas9 protein is wild-type Cas9. In embodiments of the compositions, uses, and methods, the Cas is Induces double-strand breaks in target DNA.

[0219] In some embodiments, one domain of a Cas nuclease domain protein or chimeric Cas nucleases in which regions have been replaced with parts of different proteins. In some embodiments, the Cas nuclease domain is a nucleotide sequence similar to that of Fok1. In some embodiments, the C The as nuclease may be a modified nuclease.

[0220] In other embodiments, the Cas nuclease may be derived from a Type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a nuclease that encodes a cascade of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a ScAde complex. In some embodiments, the Cas nuclease may be a Cas3 protein. It can be derived from a type II CRISPR / Cas system. In some embodiments, the Cas nuclei The ase may have RNA cleavage activity.

[0221] In some embodiments, the RNA-guided DNA binding agent has single-stranded nickase activity. that is, by cutting one DNA strand, creating a single-strand break (also known as a "nick"). In some embodiments, the RNA-guided DNA binding agent can bind to a Cas nickase. Nickases generate nicks in dsDNA, i.e., break down one end of the DNA double helix. In some embodiments, the Ca The s-nickases are characterized by one or more modifications (e.g., point mutations) in the catalytic domain. Cas nucleases in which the endonucleolytic active site is inactivated (e.g., Cas nickases and exemplary catalytic domain modifications. See, for example, U.S. Patent No. 8,889,356 for a discussion of some In some embodiments, the Cas nickase, such as the Cas9 nickase, is an inactivated form of RuvC. It has a HNH domain or an HNH domain.

[0222] In some embodiments, the RNA-guided DNA binding agent comprises a functional nuclease domain. For example, the RNA-guided DNA binder is modified to contain only one of the above-mentioned amino acids. The protein has a mutation in one of its nuclease domains that reduces its nucleic acid cleavage activity. Alternatively, the gene may be modified so as to be completely or partially deleted. In some embodiments, nickases with RuvC domains that have reduced activity are used. In some embodiments, nickases with an inactive RuvC domain are used. In this embodiment, a nickase having an HNH domain with reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0223] In some embodiments, the conserved amino acid sequence does not include the Cas protein nuclease domain. The amino acid sequence has been substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease is a RuvC nuclease domain or a RuvC The RuvC nuclease domain may contain amino acid substitutions. Exemplary amino acid substitutions in the nuclease or RuvC-like nuclease domain include D 10A (based on the S. pyogenes Cas9 protein) For example, Zetsche et al. (2015), Cell Oct 22:163 Vol. (3): pp. 759-771. In some embodiments, the Cas nuclease is Contains amino acid substitutions in the H nuclease domain or HNH-like nuclease domain Examples of HNH nuclease domains or HNH-like nuclease domains include Typical amino acid substitutions include E762A, H840A, N863A, H983A, and and D986A (based on the S. pyogenes Cas9 protein) See, for example, Zetsche et al. (2015). Typical amino acid substitutions are D917A, E1006A, and D1255A (Francis Francisella novicida U112 Cpf1(Fn Cpf1) sequence (based on UniProtKB-A0Q7Q2(CPF1_FRATN) ) are mentioned.

[0224] In some embodiments, the mRNA encoding the nickase is a target sequence containing the sense strand and The primers are provided in combination with complementary guide RNA pairs for the sense and antisense strands, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence, DSBs can be eliminated by creating nicks on opposite strands (i.e., double nicking). In some embodiments, the use of double nicking improves specificity. In some embodiments, the D When used with two separate guide RNAs targeting the NA strand, In some embodiments, a nickase is generated in the immediate vicinity of the When used with two separate guide RNAs selected to target the target DNA, A double nick is produced.

[0225] In some embodiments, the RNA-guided DNA binding agent exhibits cleavage activity and nickel cleavage activity. In some embodiments, the RNA-guided DNA binder lacks d Cas DNA-binding polypeptides. dCas polypeptides have DNA-binding activity. However, they are substantially devoid of catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. RNA-guided DNA binders lacking cleavase and nickase activities Alternatively, the dCas DNA-binding polypeptide may contain one or more modifications in the catalytic domain. The endonucleolytic activity site is inactivated by a mutation (e.g., a point mutation) Cas nucleases of the type (e.g., the Cas nucleases described above). See Patent Application Publication Nos. 2014 / 0186958 and 2015 / 0166980. sea ​​bream.

[0226] In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains. The polypeptide may be or may comprise a fusion polypeptide.

[0227] In some embodiments, the heterologous functional domain is a domain that mediates RNA-guided DNA binding to the nucleus of a cell. For example, the heterologous functional domain may be a domain that enhances transport of the drug combination. In some embodiments, the RNA-guided DNA binding agent may be a non-linear sequence (NLS). In some embodiments, the RNA-guided The DNA binder may be fused to 1 to 5 NLSs. The NA-guided DNA binder may be fused to one NLS. When the NLS is linked to the N-terminus of the RNA-guided DNA binder sequence, Alternatively, the NLS may be linked to the C-terminus. In another embodiment, the RNA-guided DNA may be inserted within the RNA-binding agent sequence. The binding agent may be fused to two or more NLSs. The idiotype DNA binder may be fused to two, three, four, or five NLSs. In some embodiments, the RNA-guided DNA binder is fused to two NLSs. In certain circumstances, the two NLSs may be the same (e.g. , two SV40 NLS), or may be different. The RNA-guided DNA binder contains two SV40 NLS sequences linked to the carboxy terminus. In some embodiments, the RNA-guided DNA binder may be fused to a sequence. may be fused to two NLSs, one linked to the N-terminus and one linked to the C-terminus In some embodiments, the RNA-guided DNA binder is fused to three NLSs. In some embodiments, the RNA-guided DNA binder may be fused to an NLS. In some embodiments, the NLS may be a monokaryotic sequence, e.g., For example, SV40 NLS, PKKKRKV (SEQ ID NO: 600), or PKKKRRV (SEQ ID NO: 601). In some embodiments, the NLS may be a bipartite sequence. The NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 60) 2). In certain embodiments, one PKKKRKV (SEQ ID NO: 600) NLS may be linked to the C-terminus of the RNA-guided DNA binder. Multiple linkers may be included in the fusion site.

[0228] In some embodiments, the heterologous functional domain is a functional domain that is a functional group ... In some embodiments, the half-life of the RNA may be improved. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. The half-life of the DNA binding agent may be decreased. In some embodiments, the heterologous functional domain comprises: It may be possible to increase the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain reduces the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain may be capable of being reduced. may act as a signal peptide for proteolysis. In some embodiments, this proteolysis may be mediated by proteolytic enzymes, e.g. For example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent is ubiquitin or polyubiquitin. In some embodiments, the ubiquitin may be modified by the addition of a ubiquitin chain. The enzyme may be a ubiquitin-like protein (UBL). Non-limiting examples include small ubiquitin-like modifiers (SUMOs), ubiquitin cross-reactive proteins, and Ubiquitin cross-reactive protein (UC RP, also known as interferon-stimulating gene-15 (interferon-stimul ated gene-15 (ISG15), ubiquitin-related modifier-1 (ubiq uitin-related modifier-1 (URM1), expressed in neural progenitor cells Developmentally downregulated neuronal precursor cell protein-8 (neuronal precursor cell protein-8) expressed developmentally downregulated protein-8) (NEDD8, in S. cerevisiae Ru b1), human leukocyte antigen F-related (human leukocyte antigen igen F-associated) (FAT10), autophagy-8 (ATG8 ) and autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB 1) Membrane-anchored UBL (MUB) , ubiquitin fold-modifying factor-1 fier-1 (UFM1), and ubiquitin-like protein-5 (UBL5). can be.

[0229] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of target domains include fluorescent proteins, purification tags, epitope tags, and In some embodiments, the marker domain includes a reporter gene sequence. The fluorescent protein may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins, Photoproteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfG FP, EGFP, Emerald, Azami Green, Monomeric Az ami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent tan Proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiY) FP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, A zurite, mKalamal, GFPuv, Sapphire, T-sapphir e), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet, Am Cyan1, Midoriishi-Cyan), red fluorescent protein (e.g., mKa te, mKate2, mPlum, DsRed monomer, mCherry, mR FP1, DsRed-Express, DsRed2, DsRed-Monomer, H cRed-Tandem, HcRed1, AsRed2, eqFP611, mRasbe rry, mStrawberry, Jred), and orange-yellow fluorescent protein (mOra nge, mKO, Kusabira-Orange, Monomeric Kusabi ra-Orange, mTangerine, tdTomato), or Any other suitable fluorescent protein may also be included. In other embodiments, the marker domain is , a purification tag and / or an epitope tag. Non-limiting exemplary tags The enzymes are glutathione-S-transferase (GST), chitin-binding protein ( CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(N ANP), tandem affinity purification (TAP) tags, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag1, Softag3 ,Strep,SBP,Glu-Glu,HSV,KT3,S,S1,T7,V5,VS VG, 6xHis, 8xHis, biotin carboxyl carrier protein (BCC Non-limiting exemplary reporters include poly-His, poly-P, and calmodulin. The target genes include glutathione-S-transferase (GST), horseradish pea peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β- Galactosidase, β-glucuronidase, luciferase, or fluorescent proteins are examples. It can be obtained.

[0230] In additional embodiments, the heterologous functional domain binds the RNA-guided DNA binder to specific cells. It may be targeted to a subcellular organelle, cell type, tissue, or organ. In this state, the heterologous functional domain targets RNA-guided DNA binders to mitochondria. It may also be possible to

[0231] In another embodiment, the heterologous functional domain may be an effector domain. When an A-guided DNA binder is directed to its target sequence, e.g., a Cas nuclease When the enzyme is directed to a target sequence by a gRNA, the effector domain acts on the target. Some embodiments may alter or affect the target sequence. In the present study, the effector domain may be a nucleic acid binding domain, a nuclease domain (e.g., a non- Cas nuclease domain), epigenetic modification domain, transcription activation domain or a transcriptional repressor factor domain. In embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, The heterologous functional domain is a transcriptional activator or a transcriptional repressor. Repurposing CRISPR as an RNA-guided plat form for sequence-specific control of ge ne expression,” Cell, vol. 152: pp. 1173–83 (2013). ;Perez-Pinera et al., “RNA-guided gene activation on by CRISPR-Cas9-based transcription fa ctors”, Nat.Methods, vol. 10: pp. 973-6 (2013);Mal i et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineer ing”, Nat. Biotechnol., vol. 31: pp. 833-8 (2013); G ilbert et al., “CRISPR-mediated modular RNA-gui ded regulation of transcription in eukar See "Cell," Cell, 154:442-51 (2013). Thus, RNA-guided DNA binding agents essentially bind to a desired target sequence using a guide RNA. This results in a transcription factor that can be induced to bind to

[0232] D. Determining gRNA Efficacy In some embodiments, the efficacy of the gRNA is assessed by comparing it with other components forming the RNP. In some embodiments, the gRNA is determined to be: It is expressed together with an RNA-guided DNA binder, such as a Cas protein (e.g., Cas9). In some embodiments, the gRNA is capable of binding to a Cas nuclease or nickase (e.g., RNA-guided DNA nucleases, such as Cas9 nuclease or nickase The gene is delivered to or expressed in a cell line that already stably expresses the enzyme. In some embodiments, the gRNA is delivered to the cell as part of an RNP. In embodiments, the gRNA is a Cas nuclease or nickase (e.g., Cas9 nuclease). mRNA encoding an RNA-guided DNA nuclease such as a nuclease or nickase It is delivered to cells together with NA.

[0233] As described herein, the RNA-guided DNA nucleases and and guide RNA to create double-strand breaks in DNA, which are then repaired by the cellular machinery. Later, errors in the form of insertion / deletion (indel) mutations can occur. Many of the mutations introduced by nucleotide sequences alter the reading frame or introduce premature stop codons, resulting in This results in a non-functional protein.

[0234] In some embodiments, the efficacy of a particular gRNA is determined based on an in vitro model. In some embodiments, the in vitro model is a HEK stably expressing Cas9. 293 cells (HEK293_Cas9). In some embodiments, The model is a peripheral blood mononuclear cell (PBMC). The primary cells are T cells, such as primary human T cells. The use of primary cells is important for consistency between experiments. In some embodiments, commercially available primary cells can be used to grow larger. , off-target deletions or insertions in in vitro models (e.g., T cells) The number of sites can be determined, for example, by the number of sites required to transfer Cas9 mRNA and guide RNA in vitro. It can be determined by analyzing genomic DNA obtained from the transfected cells. In some embodiments, such determination is performed by in vitro analysis of Cas9 mRNA, guide RNA, , and genomic DNA obtained from cells transfected with donor oligonucleotides. An exemplary procedure for such determination involves analyzing the NA. Examples in which PBMCs were used, and examples in which human CD3 + T cells were used Examples are provided.

[0235] In some embodiments, the efficacy of a particular gRNA is assessed in multiple in vitro cell models. In some embodiments, the selection step is carried out by determining the gRNA. Cell line comparisons of data using modified gRNAs are performed. In this case, cross-screening in multiple cell models is performed.

[0236] In some embodiments, the efficacy of the guide RNA is assessed by measuring the expression of TRBC1, TRBC2, and and / or TRAC indel rate. The efficacy of NA is measured by the indel rate in TRBC and / or TRAC. In some embodiments, the efficacy of the guide RNA is assessed by measuring the indel rate of TRBC or TRAC. In some embodiments, TRBC1, TRBC2, and / or The TRAC editing rate is determined by the TRBC1 protein product, the TRBC2 protein product, and and / or the indel rate and ratio required to achieve knockdown of TRAC protein product are compared.

[0237] In some embodiments, the efficacy of the guide RNA is assessed by measuring the activity of the T cell receptor (TCR) constituents. In some embodiments, the T cell receptor (TCR) is expressed as a T cell receptor (TCR), which is measured by a decrease or loss of expression of the T cell receptor (TCR). The decreased or absent expression of components of CR, such as TRAC, TRBC1, TRBC2, and CD The present invention encompasses reduction or elimination of expression of CD3E, CD3G, and / or CD3D. In this state, the reduction or disappearance of the expression of the above TCR components is One or more, for example one or two, for example one gRNA molecule described herein In each embodiment, the expression of the TCR components described above is The reduction or elimination can be measured, for example, by flow cytometry, as described herein. It is a decrease or disappearance, like

[0238] In some embodiments, the efficacy of the guide RNA is assessed by measuring the genomic DNA of a target cell type, such as a T cell. The frequency of indels is measured by the number and / or frequency of indels in off-target sequences within the target gene. In some embodiments, the indel occurrence in the cell population and / or at the target site is Off-target sites are only induced at a very low frequency (e.g., <5%) compared to the frequency of synthesis. Thus, the present disclosure provides an effective guide RNA that does not cause distortion in target cell types. Does not exhibit off-target indel formation in cells (e.g., T cells) or <5% of indels in the target region and / or compared to the frequency of indels at the target region Provide guide RNAs that result in a higher frequency of off-target indel formation. In embodiments, the present disclosure provides a method for preventing any off-target interactions with a target cell type (e.g., T cells). In some embodiments, for example, the present invention provides a guide RNA that does not exhibit nucleic acid sequence formation. Fewer than five off-target sites as assessed by one or more of the methods described in the product description In some embodiments, guide RNAs are provided that only generate indels in For example, 4, 3, 2, or 1 when assessed by one or more methods described herein. Guide RNAs are provided that generate indels only at off-target sites below the target site. In some embodiments, the off-target site is a region within the genome of a target cell (e.g., a hepatocyte). It does not occur in protein coding regions.

[0239] In some embodiments, the target DNA is subjected to insertion / deletion ("indel") mutations. Detection of gene editing events, such as gene transcription, and homologous recombination repair (HDR) events is possible using tag This utilizes linear amplification using tagged primers and isolation of tagged amplification products (hereafter referred to as LAM- This is called the "PCR" method or the "linear amplification (LA)" method.

[0240] In some embodiments, the efficacy of the guide RNA is assessed by measuring the expressed protein of the gene. In some embodiments, the level of a functional protein complex containing the protein product is measured. The efficacy of guide RNAs can be assessed by analyzing live post-edited cell populations for TCR reduction. CR expression is measured by flow cytometry analysis.

[0241] III. Methods, Including Methods of Treatment and Making Engineered Cells or Immunotherapeutic Reagents The gRNAs and related methods and compositions disclosed herein are useful in immunotherapy. and are useful in generating immunotherapeutic reagents such as engineered cells.

[0242] In some embodiments, gRNAs comprising guide sequences from Tables 1 and 2 are used in Cas nucleic acids. Together with RNA-guided DNA nucleases such as ribosomal cleavage enzymes, they induce DSBs and repair them. non-homologous ending joining (N HEJ) by TRBC1 gene, TRBC2 gene, and / or TRAC gene In some embodiments, NHEJ results in a nucleotide deletion or insertion. This results in a frameshift or nonsense mutation in the TRBC1 gene. , TRBC2 gene, and / or TRAC gene.

[0243] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a cow, a pig, a monkey, a sheep, a dog, a cat, a fish, or poultry.

[0244] In some embodiments, the preparation of a medicament for treating a human subject in need of immunotherapy. For this purpose, a guide RNA comprising any one or more of the guide sequences in Table 1 and Table 2, or or one or more sgRNAs of Table 3 (e.g., compositions provided herein). Use of the

[0245] In some embodiments, the guide RNAs, compositions, and formulations described above are used to T cells with TRBC1, TRBC2, and / or TRAC genes The modified T cells are generated ex vivo. The modified T cells may be either universal TCR or modified TCR. The T cells may express a T cell receptor, such as a CAR, or ζ It may express a CAR construct with a signaling chain motif.

[0246] In some embodiments, a single composition comprising a guide RNA provided herein A single dose was sufficient to knock down the expression of TRBC1, TRBC2, or TRAC. In other embodiments, the compositions comprising the guide RNA provided herein Two or more administrations may be beneficial to increase the therapeutic effect.

[0247] Delivery of gRNA compositions Lipid nanoparticles (LNPs) are effective in delivering cargo nucleotides and cargo proteins. and is a useful means for delivering the guide RNA, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs may be nucleic acids, proteins, or nucleic acids and proteins. delivers proteins.

[0248] A guide RNA or other nucleic acid (e.g., a nucleic acid encoding a polypeptide) is delivered to a lipid nanotube. Suitable LNPs are known in the art. For example, PCT / US2018 / 053559, International Publication No. 2017 / 17305 4, 2015 / 095340, and 2014 / 136086, as well as See the lipids references cited therein.

[0249] In some embodiments, the present invention relates to any of the gRNAs disclosed herein. a method for delivering any one of the gRNAs to a subject, wherein the gRNA is delivered via LNP; In some embodiments, the gRNA / LNP also comprises a Cas9 or or to the mRNA encoding Cas9.

[0250] In some embodiments, the present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a subject a method ... a method for the treatment of cancer, and a method for the treatment of cancer. In some embodiments, the composition comprises Cas9 or a composition comprising Cas9. It further includes the encoding mRNA.

[0251] In some embodiments, the LNP comprises an ionic lipid. The LNP is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl) (((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy) Methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octadecanoate) (Ctyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy )carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-di enoate), or another ionic lipid. See, e.g., PCT / US2018 / 053 559 (filed September 28, 2018), WO 2017 / 173054, WO 2017 / 173054, 015 / 095340 and 2014 / 136086, and the See the lipids in the cited references. In some embodiments, the positively charged cations associated with the LNP lipids The terms ionic and ionizable are interchangeable; for example, ionic lipids are It is cationic.

[0252] In some embodiments, the LNs associated with the gRNAs disclosed herein P is useful in making a medicament for treating a disease or disorder.

[0253] Electroporation is another useful means of cargo delivery and is described herein. Any electroporation method can be used to deliver any one of the disclosed gRNAs. In some embodiments, any one of the gRNAs disclosed herein may be used. , and electroporation to deliver Cas9 or mRNA encoding Cas9. Ration may also be used.

[0254] In some embodiments, the present invention relates to any of the gRNAs disclosed herein. a method for delivering any one of the gRNAs to cells in vitro, wherein the gRNA is linked to a LNP; Some embodiments include methods in which the method is linked to a nucleotide sequence or is not linked to a LNP. In this case, the gRNA / LNP or gRNA may also be Cas9 or a gene encoding Cas9. It binds to the mRNA.

[0255] In some embodiments, a single vector or vectors may be used. , the guide RNA compositions described herein are formulated in lipid nanoparticles, or and via lipid nanoparticles. See, e.g., U.S. Pat. No. 6,117,393, the entire contents of which are incorporated herein by reference. See WO 2017 / 173054, which is incorporated herein by reference.

[0256] In certain embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising any one of the guide sequences described herein. or a DNA or RNA vector encoding one of the guide RNAs containing In some embodiments, in addition to the guide RNA sequence, the vector comprises a guide RNA sequence. The nucleic acid encoding the guide RNA is also included. These include promoters, enhancers, regulatory sequences, and nuclear factors such as Cas9. Examples of nucleic acids that encode an RNA-guided DNA nuclease include those that encode an RNA-guided DNA nuclease, which may be an enzyme. In some embodiments, the vector may include, but is not limited to, crRNA, tr RNA, or one or more nucleotide sequences encoding crRNA and trRNA In some embodiments, the vector comprises an sgRNA and a Cas9 or C Encodes an RNA-guided DNA nuclease that may be a Cas nuclease, such as pf1 and one or more nucleotide sequences encoding the mRNA. In this embodiment, the vector contains crRNA, trRNA, and a Cas protein such as Cas9. an mRNA encoding an RNA-guided DNA nuclease, which may be a protein; In one embodiment, the Cas9 comprises one or more nucleotide sequences encoding the pyogenic strains. Streptococcus pyogenes (i.e., Spy Ca In some embodiments, the crRNA, trRNA, or crRNA and The nucleotide sequence encoding the guide sequence and the trRNA (which may be an sgRNA) is and all or part of the adjacent repeat sequences derived from a naturally occurring CRISPR / Cas system. and comprising or consisting of crRNA, trRNA, or crRNA and A nucleic acid comprising or consisting of a trRNA is a nucleic acid that naturally contains the crRNA, trRNA, or A, or containing or from nucleic acids not present together with crRNA and trRNA It may further comprise a vector sequence.

[0257] In some embodiments, the components can be introduced as naked nucleic acid or It can also be delivered as a liposome or nucleic acid complexed with an agent such as poloxamer. Alternatively, nucleic acids (e.g., gRNA) and ribonucleoprotein complexes (RNPs) may be used. In some embodiments, the components can be introduced as a protein. Viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus) The nucleic acid may also be delivered by a vector, such as a non-viral vector (e.g., a serotype vector, a lentivirus, etc.). Methods and compositions for delivery include electroporation, lipofection, Microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, LNP , polycation or lipid:nucleic acid complexes, naked nucleic acids (e.g., naked DNA / RNA), human These include the production of virions and the enhancement of DNA uptake by drugs. Sonoporation using the 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

[0258] The introduction of a polypeptide into a cell involves the use of a nucleic acid template or other nucleic acid encoding the polypeptide of interest. This can be achieved by delivering a nucleic acid to the cell. may optionally be encoded as part of an expression cassette, in which case the template is Optionally, such flanking sequences are present in the template in the cell. With homology arms designed to promote homologous recombination repair and integration of nucleic acids The nucleic acid template contains an oligonucleotide sequence encoding a polypeptide of interest flanked by sequences homologous to the first target locus. By including an open reading frame (ORF), homologous recombination at the target locus The template may contain homology arms flanking the gRNA cleavage site. For example, the homology arms may comprise flanking sequences. The flanking sequences exclude the region of the target locus at or around the cleavage site. This deletion prevents cleavage at this site after repair and also prevents the gRNA / Cas from The gRNA cleavage site is designed to prevent the gRNA from cleaving the template nucleic acid. The outer regions or "gaps" are 5, 10, 20, 30, 40, 50, 60, 70, 80, The flanking sequences may be 90, 100, 150, or more nucleotides in length. For example, gaps of 10 to 100 nucleotides, gaps of 10 to 50 nucleotides, 2 Remove gaps between 5 and 100 nucleotides or gaps between 50 and 100 nucleotides It can be designed to:

[0259] The nucleic acid encoding the polypeptide of interest is contained in a viral or non-viral template. The viral vector may be any suitable virus, for example, a retrovirus. Rovirus, adenovirus, lentivirus, adeno-associated virus, or any of these viruses In some embodiments, the viral vector is an adeno-associated virus. The nucleic acid template may be an AAV vector. The endogenous TRAC promoter may also be included in the target polypeptide after insertion. The nucleic acid template may be promoterless, since it can drive expression.

[0260] In addition to the sequence encoding the polypeptide of interest, the nucleic acid template may contain a promoter sequence, a Kozak sequence, The sequence includes the IRES sequence, splice acceptor sequence, polyA sequence, and P2A and T2 and one or more of the sequences encoding cleavable peptides such as E2A, E3A, E4A, E5A, E6A, E7A, E8A, E9A, E10A, E11A, E12A, E13A, E14A, E15A, E16A, E17A, E18A, E19A, E20A, E21A, E22A, E23A, E24A, E25A Good too.

[0261] In some embodiments, the methods described herein comprise the step of: and delivering to a cell one or more sequences comprising one or more genes encoding the In some embodiments, the polypeptide of interest is a polypeptide involved in the regulation of immune tolerance. In some embodiments, the polypeptide of interest is involved in the regulation of a cancer phenotype. It is something that is involved.

[0262] In some embodiments, the one or more polypeptides include a receptor, e.g., Examples include immunoreceptors. means a receptor that can recognize an antigen. Exemplary types of immune receptors include T cell receptors and In some embodiments, the above-mentioned receptors are selected from the group consisting of TCRs and chimeric antigen receptors (CARs). The immune receptor recognizes a cancer antigen such as WT1. In certain embodiments, The target polypeptide is WT1 TCR. The WT1 TCR is a VLD It may be one that recognizes the FAPPGA(VLD)WT1 peptide, or RM It may also be a substance that recognizes the FPNAPYL(RMF)WT1 peptide. In this embodiment, the WT1 TCR recognizes a VLD peptide. The polypeptide may comprise the α chain of WT1 TCR. The peptide may comprise the β chain of WT1 TCR. The target polypeptide contains the WT1 TCR α chain and the WT1 TCR β chain. Exemplary WT1-specific TCR sequences are known in the art. , including the following: [Table 4] TIFF2025138662000021.tif177118JPEG2025138662000022.jpg249158JPEG2025138662000023.jpg247158JPEG20251386620 00024.jpg247158JPEG2025138662000025.jpg247158TIFF2025138662000026.tif179118TIFF2025138662000027.tif137118

[0263] Complementarity-determining regions (CDRs) are present within the variable domains of TCRs and antibodies. Each variable region of TCRα and TCRβ has three CDRs. The exact boundaries of these CDRs are determined by known methods. The sequences can be determined according to the method described in, for example, Kabat et al., "Sequences of Proteins of Immunological Interest" and D unbar et al., “ANARCI: antigen receptor numberi (See "Method and receptor classification" below.) Residue numbers that apply not only to the variable domain but also to the residue boundaries that define the three CDRs Other CDR regulations may overlap with Kabat-based CDRs. Furthermore, specific residues or groups of residues or the entire CDR do not significantly affect antigen binding. It is possible to obtain CDRs that are shortened or extended based on predictions or experimental results that The CDRs herein can be defined according to any of these methods.

[0264] The one or more sequences above may be TRAC, TRBC1, and / or TRBC2, etc. In some embodiments, the gene locus of One or more of the above genes can be inserted, e.g., integrated, into the TRAC locus. The one or more genes may be mediated by a heterologous promoter, such as the EF-1α promoter. Alternatively, one or more of the above genes may be provided in a form operably linked to the gene. The gene is expressed by the endogenous promoter after integration, e.g., the TRAC promoter, TRBC1 promoter, or in a configuration such that it is expressed from the TRBC2 promoter. The one or more genes may be composed of two or more polypeptides linked by a self-cleaving sequence. This approach can involve translational fusion of a multi-chain receptor (e.g., a T cell receptor). A coding sequence is provided and a single promoter (endogenous) is used to drive expression of the receptor. This can be useful when it is desired to use a single gene (whether native or heterologous). The activity of the promoter causes the polypeptides to be cleaved to give the individual receptor chains. The resulting mRNA contains a continuous coding sequence containing the amino acids of multiple receptor chains. do.

[0265] The sequence or sequences may be delivered by any suitable means, for example by transfer. ction, lipid nanoparticles, electroporation, microinjection, or In some embodiments, the vector is a viral vector, such as an adeno-associated viral vector. Viral vectors are used.

[0266] IV. Additional Exemplary Embodiments The following additional exemplary embodiments are provided. Embodiment 1. A method for modifying the DNA sequence within the TRBC1 gene and / or the TRBC2 gene. So, delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 2. A method for reducing expression of the TRBC1 gene and / or the TRBC2 gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 3. 1. A method of immunotherapy comprising: administering the composition to a subject, their autologous cells, and / or allogeneic cells; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 4. 1. A method for modifying a DNA sequence within a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 5. 1. A method for reducing expression of a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 6. 1. A method of immunotherapy comprising: administering the composition to a subject, their autologous cells, and / or allogeneic cells; The composition comprises: a. A guide RNA comprising a sequence selected from the following: i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a). c. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A method comprising: Embodiment 7. DNA sequences within the TRBC1 gene, TRBC2 gene and / or TRAC gene A method of modifying, A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and delivering a nucleic acid encoding an A-binding agent or an RNA-guided DNA-binding agent. , The first guide RNA is i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; and The second guide RNA is i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. A guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method of claim 1, comprising a sequence selected from Embodiment 8. Reduces the expression of TRBC1, TRBC2 and / or TRAC genes A method of A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and delivering a nucleic acid encoding an A-binding agent or an RNA-guided DNA-binding agent. , The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; and a sequence selected from The second guide RNA is i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. A guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method of claim 1, comprising a sequence selected from Embodiment 009. 1. A method of immunotherapy comprising: administering the composition to a subject, to autologous cells, or to allogeneic cells; The composition comprises a first guide RNA, a second guide RNA, and optionally an RNA guide and a nucleic acid encoding a DNA-binding agent or an RNA-guided DNA-binding agent, The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. A guide sequence comprising any one of SEQ ID NOs: 1 to 6; and a sequence selected from The second guide RNA is i. a guide sequence selected from SEQ ID NOs: 90-178; ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 guide sequence with % identity; iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; and v. A guide sequence comprising any one of SEQ ID NOs: 90 to 95; The method of claim 1, comprising a sequence selected from Embodiment 010. An RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent is administered. 10. The method according to any one of embodiments 1 to 9, wherein Embodiment 011. a. A guide RNA comprising: i. a guide sequence selected from SEQ ID NOs: 1-89; or ii. At least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; or iii. At least 99%, at least At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% a guide sequence with identity; or iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; or v. a guide sequence comprising any one of SEQ ID NOs: 1 to 6; and, optionally, b. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A composition comprising: Embodiment 012. Modifying the DNA sequence within the TRBC1 gene and / or TRBC2 gene in cells 12. The composition of embodiment 11, for use in Embodiment 013. When introducing the expression of the TRBC1 gene and / or the TRBC2 gene into cells 12. The composition of embodiment 11, wherein Embodiment 014. a. A guide RNA comprising: i. a guide sequence selected from SEQ ID NOs: 90-178; or ii. At least 17 sequences selected from SEQ ID NOs: 90 to 178, at least 1 8, at least 19, or at least 20 consecutive nucleotides; or iii. At least 99% of the sequences selected from SEQ ID NOs: 90 to 178 at least 98%, at least 97%, at least 96%, at least 95%, at least 9 4%, at least 93%, at least 92%, at least 91%, or at least 90 % identity to a guide sequence; or iv. a guide sequence comprising any one of SEQ ID NOs: 90-113; or v. a guide sequence comprising any one of SEQ ID NOs: 90-95; and, optionally, the law of nature b. an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent , A composition comprising: Embodiment 015. Embodiment 14, used in modifying a DNA sequence within a TRAC gene in a cell. The composition described in Embodiment 016. 15. The method of claim 14, wherein the method is used to reduce the expression of the TRAC gene in a cell. Composition of. Embodiment 017. The composition of any one of embodiments 11 to 17 for use in immunotherapy of a subject. Embodiment 018. A cell modified by the method according to any one of embodiments 1 to 10. Embodiment 019. 20. The cell of embodiment 18, which has been modified in vitro. Embodiment 020. 20. The cell of embodiment 18 or embodiment 19, which is a T cell. Embodiment 021. CD3 + , CD4 + and / or CD8 + Any of embodiments 18 to 20, wherein the T cell is a T cell. The cell described in any one of claims 1 to 4. Embodiment 022. Any one of embodiments 18-21, wherein the cell is a mammalian cell, a primate cell, or a human cell. The cell described in Embodiment 023. 22. The cell according to any one of embodiments 18 to 21, for use in immunotherapy of a subject. Embodiment 024. a. TRBC1 gene, TRBC2 gene and / or cells in a subject or introducing a double-strand break (DSB) within the TRAC gene; or b. TRBC1 gene, TRBC2 gene and / or cells in a subject or introducing a single-strand break (SSB) within the TRAC gene; or c. TRBC1 gene, TRBC2 gene and / or cells in a subject or reducing the expression of the TRAC gene; 11. The method of any one of embodiments 1 to 10, further comprising: Embodiment 025. The composition results in editing of the TRBC1 gene and / or the TRBC2 gene. The method or composition used in any of embodiments 1 to 3 and embodiments 7 to 13. Embodiment 026. Embodiments 4-10 and embodiment 14, wherein the composition effects editing of the TRAC gene. 17. A method or composition used in any of claims 1 to 17. Embodiment 027. Embodiments 7-1, wherein the composition results in editing of the TRBC gene and the TRAC gene. 10. The method or composition used in any of Embodiment 028. The edits are calculated as a percentage of the population that is edited (edit rate or indel rate). 28. The method or composition of any of claims 25 to 27. Embodiment 029. The editing rate is 10 to 100% of the population, for example, 30 to 99% of the population. , The method or composition used in any of embodiments 25 to 28. Embodiment 030. the editing rate is 30 to 35%, 35 to 40%, 40 to 45%, or 45 to 50% of the population; 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80 %, 80-85%, 85-90%, 90-95%, or 95-99%. 25-29. A method or composition used in any one of claims 25-29. Embodiment 031. The composition comprises: a. Any one of SEQ ID NOs: 179-184; or b. a guide sequence selected from any one of SEQ ID NOs: 1-89; or c. a guide sequence selected from SEQ ID NOs: 1-24; or d. a guide sequence selected from SEQ ID NOs: 1-6; Any of embodiments 1 to 3, 7 to 13, 17, and 24 to 30, comprising an sgRNA comprising 10. The method or composition according to claim 1 . Embodiment 032. The composition comprises: a. a guide sequence selected from any one of SEQ ID NOs: 90-178; or b. a guide sequence selected from SEQ ID NOs: 90-113; or c. a guide sequence selected from SEQ ID NOs: 90-95; any of embodiments 4 to 10, 14 to 17, and 24 to 30, comprising an sgRNA comprising 10. The method or composition according to claim 1. Embodiment 033. The target sequence is a sequence of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. Sequences within the first, second, third, or fourth exons 33. The method or composition of any one of aspects 1-17 and 24-32. Embodiment 034. The target sequence is a TRBC1 gene, a TRBC2 gene, and / or a TRAC gene. 34. The method or composition of embodiment 33, wherein the sequence is within the first exon of Embodiment 035. The target sequence is a TRBC1 gene, a TRBC2 gene, and / or a TRAC gene. 34. The method or composition of embodiment 33, wherein the sequence is within the second exon of Embodiment 036. The target sequence is a TRBC1 gene, a TRBC2 gene, and / or a TRAC gene. 34. The method or composition of embodiment 33, wherein the sequence is within the third exon of Embodiment 037. The target sequence is a TRBC1 gene, a TRBC2 gene, and / or a TRAC gene. 34. The method or composition of embodiment 33, wherein the sequence is within the fourth exon of Embodiment 038. The target sequence is selected from the group consisting of the human TRBC1 gene, the human TRBC2 gene, and / or the human TRBC3 gene. 38. The method or composition of any one of embodiments 33 to 37, wherein the sequence is within the AC gene. thing. Embodiment 039. The guide sequence is a target sequence within the positive strand of TRBC1, TRBC2, and / or TRAC. 39. The method of any one of embodiments 1 to 17 or 24 to 38, wherein the sequence is complementary to Method or composition. Embodiment 040. The guide sequence is selected from the group consisting of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. 10. The method of claim 1, wherein the target sequence is complementary to a target sequence in the negative strand of the nucleic acid sequence. 10. The method or composition of any one of claims 1 to 9. Embodiment 041. The first guide sequence is selected from the group consisting of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene. The composition is complementary to a first target sequence in the positive strand of the TRBC1 gene. , against a second target sequence within the minus strand of the TRBC2 gene and / or the TRAC gene 41. Any of embodiments 1-17 or 24-40, further comprising a second guide sequence complementary to 10. The method or composition according to claim 1. Embodiment 042. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178. and further comprising the nucleotide sequence of SEQ ID NO: 200, In embodiments 1-17 or 24, a nucleic acid sequence follows the 3' end of the guide sequence. 41. The method or composition of any one of claims 1 to 41. Embodiment 043. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178. and further comprising the nucleotide sequence of SEQ ID NO: 201, In embodiments 1-17 or 24, a nucleic acid sequence follows the 3' end of the guide sequence. 41. The method or composition of any one of claims 1 to 41. Embodiment 044. The guide RNA is modified according to the pattern of SEQ ID NO: 300, and N is and any one of the guide sequences in Table 1 (SEQ ID NOs: 1 to 89) according to embodiments 1 to 17. or 24 to 43, a method or composition according to any one of claims 24 to 43. Embodiment 045. each N in SEQ ID NO: 300 is any natural or non-natural nucleotide; The N forms a guide sequence, and the guide sequence directs Cas9 to the TRBC1 gene, TRBC 45. The method of embodiment 44, wherein the method targets the IL-2 gene and / or the TRAC gene. composition. Embodiment 046. The sgRNA is at least 99% specific to a sequence selected from SEQ ID NOs: 1 to 89, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, or at least 9 45. The method of any one of embodiments 42 to 44, comprising a guide sequence having 0% identity to the target gene. Method or composition. Embodiment 047. 1 to 17 or 24 to 46, wherein the guide RNA comprises at least one modification 10. The method or composition of any one of claims 1 to 9. Embodiment 048. wherein the at least one modification is a 2'-O-methyl (2'-O-Me) modified nucleotide 48. The method or composition of embodiment 47, comprising: Embodiment 049. 47 or 48, which comprises phosphorothioate (PS) bonds between nucleotides. 49. The method or composition of claim 48. Embodiment 050. 50. Any of embodiments 47 to 49, comprising 2'-fluoro (2'-F) modified nucleotides. 10. The method or composition according to claim 1. Embodiment 051. a modification in one or more of the first five nucleotides at the 5' end of the guide RNA; , The method or composition of any one of embodiments 47 to 50. Embodiment 052. The guide RNA contains a modification in one or more of the last five nucleotides at the 3' end. , The method or composition of any one of embodiments 47 to 51. Embodiment 053. 47. A method according to claim 46, further comprising the step of: 52. A method or composition according to any one of claims 1 to 52. Embodiment 054. 47. A method according to claim 46, further comprising the step of: 53. A method or composition according to any one of claims 1 to 53. Embodiment 055. The first three nucleotides of the 5' end of the guide RNA are 2'-O-Me modified nucleotides. 55. The method or composition of any one of embodiments 47 to 54, comprising an oxalate. Embodiment 056. The last three nucleotides at the 3' end of the guide RNA are 2'-O-Me modified nucleotides. 56. The method or composition of any one of embodiments 47 to 55, comprising an oxalate. Embodiment 057. 57. The method of claim 47, wherein the guide RNA comprises modified nucleotides of SEQ ID NO: 300. 10. The method or composition of any one of claims 1 to 9. Embodiment 058. 17. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient. 24-57. A method or composition according to any one of claims 24 to 57. Embodiment 059. Embodiments 1-58, wherein the guide RNA is associated with a lipid nanoparticle (LNP). 10. The method or composition of any one of claims 1 to 9. Embodiment 060. 60. The method or composition of embodiment 59, wherein said LNP comprises a biodegradable ionic lipid. . Embodiment 061. The ionic lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butano yl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy )methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis( octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy) (9Z,12Z)-octadeca-9,12-carbonyloxymethylpropyl 61. The method or composition of embodiment 60, wherein the compound is a dienoate. Embodiment 062. 62. The method of any one of embodiments 59-61, wherein the LNPs comprise neutral lipids. composition. Embodiment 063. 63. The method or composition of embodiment 62, wherein said neutral lipid is DSPC. Embodiment 064. 64. The method or method of any one of embodiments 59-63, wherein said LNP comprises a helper lipid. Or composition. Embodiment 065. 65. The method or composition of embodiment 64, wherein said helper lipid is cholesterol. Embodiment 066. Embodiments 59-6, wherein the LNP comprises a stealth lipid 6. The method or composition of any one of 5. Embodiment 067. 67. The method or composition of embodiment 66, wherein the stealth lipid is PEG2k-DMG. thing. Embodiment 068. 25. Embodiments 1-17 or 24, wherein the composition further comprises an RNA-guided DNA binding agent. 67. A method or composition according to any one of claims 1 to 66. Embodiment 069. In one embodiment, the composition further comprises an mRNA encoding an RNA-guided DNA-binding agent. 69. The method or composition according to any one of aspects 1 to 17 or 24 to 68. Embodiment 070. Embodiment 68 or embodiment 69, wherein the RNA-guided DNA binding agent is Cas9. 2. The method or composition according to claim 1 . Embodiment 071. Embodiments 1-1, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier. 7 or 24-70. The method or composition according to any one of claims 7 or 24-70. Embodiment 072. Embodiments 1 to 3, 7 to 1, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 1. The method, composition, or method according to any one of claims 3, 17, 24, 25, 27 to 31, or 33 to 71. A substance or cell. Embodiment 073. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 2. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 074. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 3. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 075. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 4. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 076. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 5. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 077. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 6. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 078. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 7. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 079. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 8. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 080. In embodiments 1 to 3, 7 to 8, the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 9. 13, 17, 24, 25, 27 to 31, or 33 to 71. Or composition. Embodiment 081. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 10. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 082. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 11. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 083. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 12. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 084. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 13. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 085. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 14. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 086. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 15. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 087. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 16. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 088. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 17. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 089. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 18. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 090. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 19. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 091. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 20. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 092. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 21. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 093. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 22. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 094. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 23. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 095. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 24. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 096. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 25. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 097. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 26. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 098. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 27. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 099. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 28. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 100. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 29. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 101. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 30. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 102. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 31. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 103. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 32. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 104. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 33. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 105. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 34. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 106. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 35. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 107. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 36. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 108. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 37. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 109. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 38. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 110. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 39. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 111. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 40. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 112. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 41. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 113. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 42. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 114. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 43. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 115. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 44. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 116. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 45. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 117. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 46. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 118. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 47. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 119. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 48. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 120. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 49. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 121. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 50. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 122. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 51. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 123. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 52. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 124. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 53. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 125. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 54. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 126. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 55. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 127. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 56. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 128. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 57. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 129. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 58. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 130. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 59. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 131. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 60. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 132. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 61. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 133. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 62. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 134. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 63. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 135. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 64. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 136. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 65. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 137. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 66. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 138. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 67. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 139. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 68. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 140. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 69. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 141. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 70. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 142. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 71. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 143. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 72. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 144. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 73. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 145. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 74. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 146. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 75. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 147. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 76. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 148. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 77. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 149. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 78. 13, 17, 24, 25, 27-31, or 33-71. Or composition. 150. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 79. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 151. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 80. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 152. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 81. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 153. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 82. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 154. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 83. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 155. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 84. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 156. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 85. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 157. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 86. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 158. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 87. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 159. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 88. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 160. Embodiments 1 to 3, 7, wherein the sequence selected from SEQ ID NOs: 1 to 89 is SEQ ID NO: 89. 13, 17, 24, 25, 27-31, or 33-71. Or composition. Embodiment 161. Embodiments 4 to 10, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 90. 14 to 17, 24, 26 to 30, or 32 to 71, or composition. Embodiment 162. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 91. 24, 26-30, or 32-71. is a composition. Embodiment 163. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 92 24, 26-30, or 32-71. is a composition. Embodiment 164. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 93 24, 26-30, or 32-71. is a composition. 165. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 94. 24, 26-30, or 32-71. is a composition. Embodiment 166. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 95. 24, 26-30, or 32-71. is a composition. Embodiment 167. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 96 24, 26-30, or 32-71. is a composition. Embodiment 168. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 97 24, 26-30, or 32-71. is a composition. Embodiment 169. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 98 24, 26-30, or 32-71. is a composition. Embodiment 170. Embodiments 4 to 1, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 99 24, 26-30, or 32-71. is a composition. Embodiment 171. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 100. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 172. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 101. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 173. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 102. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 174. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 103. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 175. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 104. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 176. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 105. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 177. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 106. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 178. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 107. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 179. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 108. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 180. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 109. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 181. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 110. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 182. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 111. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 183. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 112. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 184. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 113. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 185. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 114. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 186. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 115. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 187. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 116. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 188. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 117. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 189. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 118. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 190. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 119. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 191. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 120. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 192. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 121. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 193. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 122. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 194. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 123. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 195. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 124. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 196. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 125. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 197. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 126. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 198. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 127. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 199. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 128. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 200. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 129. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 201. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 130. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 202. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 131. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 203. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 132. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 204. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 133. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 205. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 134. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 206. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 135. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 207. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 136. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 208. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 137. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 209. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 138. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 210. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 139. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 211. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 140. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 212. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 141. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 213. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 142. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 214. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 143. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 215. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 144. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 216. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 145. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 217. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 146. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 218. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 147. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 219. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 148. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 220. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 149. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 221. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 150. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 222. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 151. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 223. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 152. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 224. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 153. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 225. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 154. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 226. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 155. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 227. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 156. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 228. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 157. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 229. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 158. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 230. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 159. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 231. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 160. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 232. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 161. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 233. Embodiment 4 to 5, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 162. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 234. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 163. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 235. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 164. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 236. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 165. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 237. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 166. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 238. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 167. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 239. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 168. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. 240. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 169. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 241. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 170. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 242. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 171. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 243. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 172. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 244. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 173. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 245. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 174. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 246. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 175. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 247. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 176. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 248. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 177. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. Embodiment 249. Embodiment 4 to 178, wherein the sequence selected from SEQ ID NOs: 90 to 178 is SEQ ID NO: 178. 10, 14 to 17, 24, 26 to 30, or 32 to 71. Or composition. 250. The composition, formulation, or is the use of cells. Embodiment 251. The composition, formulation, or Or cells. Embodiment 252. To generate T cells that lack endogenous T cell receptors and express non-endogenous T cell receptors. The cell according to any one of embodiments 18 to 23 for use in a method for producing a cell comprising the steps of: Embodiment 253. To generate CAR-expressing T cells that lack endogenous T cell receptors, 24. The cell of any one of aspects 18 to 23. Embodiment 254. After modification, CD3 - The cell according to any one of embodiments 18 to 23, which is a cell. Embodiment 255. Before modification, it was CD3 + cells, and after modification, CD3 - Any of embodiments 18 to 23, which becomes a cell. A cell described in any one of claims 1 to 4. Embodiment 256. A cell population comprising the cells according to any one of embodiments 18 to 23, wherein the cell population is a modified population. More than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80% > 85%, > 90%, > 95%, > 98%, or > 99% CD3 - A cell, a group of cells. Embodiment 257. Approximately 90% of the population is CD3 -257. The population of embodiment 256, wherein Embodiment 258. Approximately 95% of the population is CD3 - 257. The population of embodiment 256, wherein 259. Approximately 99% of the population is CD3 - 257. The population of embodiment 256, wherein 260. A cell population comprising the cells of any one of embodiments 18 to 23, wherein about 5% of the population More than 0%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, More than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of endogenous T cell receptors A cell population that lacks a signaling pathway. 261. 261. The population of embodiment 260, wherein more than about 90% of the population lacks an endogenous T cell receptor. . Embodiment 262. 261. The population of embodiment 260, wherein more than about 95% of the population lacks an endogenous T cell receptor. . Embodiment 263. 261. The population of embodiment 260, wherein greater than about 99% of the population lacks endogenous T cell receptors. . Embodiment 264. The occurrence of the TRBC1, TRBC2 and / or TRAC genes in the population The expression is at least about 50%, at least about 55%, or less than that of the same unmodified cell population. at least about 60%, at least about 65%, at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95%, Any of embodiments 18-23, wherein the amount of erythrocytes is reduced by at least about 98%, or by at least about 99%. A cell population comprising the cells described in any one of claims 1 to 4. 265. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 265. The population of embodiment 264, wherein the population is at least about 90%. Embodiment 266. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 265. The population of embodiment 264, wherein the population is at least about 95%. Embodiment 267. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 265. The population of embodiment 264, wherein the population is at least about 99%. Embodiment 268. Any of embodiments 264 to 267, wherein the decrease is a decrease in the expression of the TRBC1 gene. The population described in paragraph 1. 269. Any of embodiments 264 to 267, wherein the decrease is a decrease in the expression of the TRBC2 gene. The population described in paragraph 1. 270. Any one of embodiments 264 to 267, wherein the decrease is a decrease in expression of the TRAC gene. The population described in section . 271. A cell population comprising the cells according to any one of embodiments 18 to 23, 10 to 100%, for example, 30 to 99% of the population, A cell population with indels in the IFN-γ and / or TRAC genes. Embodiment 272. 30-35%, 35-40%, 40-45%, 45-50%, 50-55% of the population , 55~60%, 60~65%, 65~70%, 70~75%, 75~80%, 80~8 5%, 85-90%, 90-95%, or 95-99% of the TRBC1 gene, TRBC 272. The method of claim 271, wherein the fusion protein of claim 271 has an indel in the TRAC gene and / or the TRAC gene. Group. Embodiment 273. 271 or embodiment, wherein the indel is in the TRBC1 gene. 272. The population described in Embodiment 274. 271 or embodiment, wherein the indel is in the TRBC2 gene. 272. The population described in 275. Embodiment 271 or embodiment 2, wherein the indel is in the TRAC gene. 72. The population described in 72.

[0267] This description and the exemplary embodiments should not be considered limiting. All numbers expressing amounts, percentages, or ratios used in the claims and and other numerical values ​​are understood in all cases to be by the term "about," unless otherwise noted. To the extent that it is not so modified, it should be understood to be modified. , unless otherwise specified, the numerical values ​​set forth in the following specification and appended claims. The parameters are approximate and may vary depending on the desired properties sought to be obtained. and is not intended to limit the application of the doctrine of equivalents to the scope of the claims. Instead, each numerical parameter should at least be reported to the nearest significant digit and using ordinary rounding techniques. should be interpreted in accordance with the

[0268] As used in this specification and the appended claims, the singular forms "a," "a" and "b" are used interchangeably. Any use of "n" and "the" and any singular form of any word is express and clear. Note that unless a statement is limited to a single referent, it encompasses multiple referents. As used herein, the term "include" and its grammatical variations The phraseology is intended to be non-limiting, and the description of an item within a list is indicative of the specific item being listed. It does not exclude other similar items that may be substituted or added. [Example]

[0269] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of the present disclosure. It does not limit the scope of the disclosure in any way.

[0270] Example 1: Materials and Methods Genomic DNA isolation HEK293_Cas9 transfected cells 24 hours after transfection 50 μL / well of QuickExtract™ DNA Extractor Action Solution (Lucigen, Catalog Number QE 09050) according to the manufacturer's protocol from each well of a 96-well plate. gDNA was extracted from the samples. DNA samples were subjected to PCR as described herein. Then, NGS analysis was performed.

[0271] Next-generation sequencing ("NGS") and on-target cleavage efficiency analysis Deep sequencing is used to quantify the efficiency of editing at targeted locations in the genome. The presence of insertions and deletions introduced by gene editing was identified. PCR primers were designed around the target site within the nucleotide sequence AC to amplify the genomic region of interest. The primer sequences were designed according to standard methods in the art.

[0272] To add sequencing chemistry, the manufacturer (Illumina) An additional PCR was performed according to the protocol of ). The amplicons were purified using Illumina MiSe After filtering out reads with low quality scores, the reads were aligned to the human reference genome ( The reads were aligned against a target region of interest (e.g., hg38). The number of wild-type reads versus the number of reads containing insertions or deletions ("indels") was calculated. .

[0273] The editing rate (e.g., "editing efficiency" or "indel rate") is the rate at which insertions or deletions ("indels") occur. The total number of sequence reads with wild-type variance (i.e., "wild-type") is defined as the total number of sequence reads with wild-type variance (i.e., "wild-type") divided by the total number of sequence reads with wild-type variance (i.e., "wild-type")

[0274] Example 2: Design of TRAC guides and screening in HEK cells Design of the human TRAC guide Human reference genome (e.g., hg38) and user-defined genomic regions of interest (e.g., Initial guide selection in silico using exons encoding the TRAC protein For each PAM identified, The analysis was performed and the statistics reported. Several evaluation criteria known in the art (e.g., Based on the GC content, predicted on-target activity, and potential off-target activity, Further selection and ranking of the gRNA molecules was performed.

[0275] TRAC (ENSG00000277734) A total of 88 guide RNAs were designed targeting the intron region and intron 1. The id sequences and corresponding genome coordinates are shown (Table 2).

[0276] Guides were screened based on their editing efficiency in HEK293_Cas9 cells. The human embryonic kidney adenocarcinoma cell line HEK293 (HEK293) constitutively expresses SpyCas9. The cells (293_Cas9) were cultured in DMEM medium supplemented with 10% fetal bovine serum. The cells were seeded in a 96-well plate at a density of 10,000 cells / well, and transfected after approximately 24 hours. Transfection was performed (approximately 70% confluent at the time of transfection). Cell transfection was performed in duplicate using Lipofectamine RNAiMAX (ThermoFisher, catalog number 1 3778150) according to the manufacturer's protocol. M), trRNA (25nM), Lipofectamine RNAiMAX (0.3 Lipoplexes containing 10 μL / well of OptiMem were used to transfect cells. DNA isolation and NGS analysis were performed as described in Example 1. and Table 5 shows the TRAC gene expression profiles of these guides in HEK293_Cas9 cells. The indel rate at each locus is shown.

[0277] [Table 5] TIFF2025138662000029.tif115170

[0278] Example 3: Human CD3 + TRAC-guided screening in T cells

[0279] The indel editing rate in the HEK293_Cas9 cells obtained in Example 2 was the highest. The 24 guides were then combined with human CD3 + Editing efficiency and T cell receptor (TC) R) Screening based on knockdown of CD3 +T cells are CD4 + helper T cells and CD8 + It is composed of multiple T cell populations, including cytotoxic T cells. T cells can be isolated from whole blood or leukapheresis samples. By engineering parental T cells with Cas9-mediated editing, cancer cells can be specifically targeted. It can be engineered to be targeted and less immunogenic. In T cells, Cas9 RNP (e.g., Cas9 RNP targeting TRAC) The basic method used to deliver NPs is described. This protocol can be used to To match a cellular target (e.g., any of those provided herein), RN Only the targeting crRNA of P needs to be changed.

[0280] Delivery of RNP to T cells T cells can be obtained commercially (e.g., Human Peripheral Blood CD4 + CD45RA + T Cells, Frozen, Stem Cell Technology, Inc. Stem Cell Technology, catalog number 70029) or For in-house preparation, the commercial kit was first prepared. (e.g., EasySep™ Human T Cell Isolation Enrich T cells from Leukopak using a kit (Stem Cell Technology, Inc.) The enriched T cells were aliquoted and frozen (5 × 10 6 pcs / bya Afterwards, the vial was thawed as needed and T cell medium (RPMI1640, FB S, L-glutamine, essential amino acids, sodium pyruvate, HEPES buffer, 2-methyl- CD3 / CD28 beads in a 3:1 ratio in 100 ml of HCl (100 ml of HCl, ... RNP was activated by adding Dynabeads (Life Technologies). The individual samples were prepared by mixing equal volumes of the reagents, incubating at 95°C for 2 minutes, and then cooling to room temperature. It was prepared by preannealing individual crRNAs and trRNAs. The dual guide (dgRNA) consisting of the crRNA and trRNA was then introduced into the Spy Ca Incubation with s9 protein results in the formation of ribonucleoprotein (RNP) complexes Spy Cas9 (10 nM), individual guides (10 nM), and tracers RNP containing RNA (10 nM) was cultured in P3 Primary Cell 96-we ll Nucleofector™ Kit (Lonza, catalog no. The plate was placed in an Amaxa™ 96-well S Huttle(TM) Protocol for Stimulated Human By nucleofection with CD3 T cells + Tryptophan T cells Immediately after nucleofection, T cell medium was added to the cells. The mixture was added and cultured for 2 days or more.

[0281] Two days after nucleofection, genomic DNA was prepared as described in Example 1 and analyzed by NG Table 6 and Figure 2 show the CD3 + TRAC by various guides in T cells Results for indel frequency after editing are shown.

[0282] [Table 6]

[0283] Flow cytometry analysis of TCR expression Four days after nucleofection, TCR expression was measured. The TCR was stained with a standard dye (Thermo Fisher L34975) and Alexa Flu or(registered trademark)647 anti-human TCR α / β Antibody( Detection was performed using BioLegend (BioLegend, Cat. No. 306714). Cells were incubated with 2 μl of antibody for at least 20 min on ice and then purified by Beckman Cytoflex S manufactured by Beckman Coulter and other similar devices were used. Flow cytometry was used to analyze the data. The viable cell population was analyzed for TCR reduction. The results are shown in Table 7 and Figure 3.

[0284] [Table 7]

[0285] Example 4: Design of TRBC guides and screening in HEK cells Design of human TRBC guide Human TRBC1 (ENSG00000211751) and TRBC2 (ENSG00 The initial guide selection for editing (000211772) was performed as described in Example 2. A total of 89 guide RNAs were designed: two that target only TRBC1 (SEQ ID NO: 1); 11 and SEQ ID NO: 26), two species that target only TRBC2 (SEQ ID NO: 8), and T 86 species targeting both RBC1 and TRBC2. Guides are protein exon codes. The guides and corresponding genomic coordinates are listed above. As shown in Table 1.

[0286] TRBC guides were screened based on editing efficiency in HEK293_Cas9 cells. Transfection was performed as described in Example 2. DNA isolation and NGS analysis was performed as described in Example 1. Figures 4A and 4B and Table 8 show the HE These guides transfected the TRBC1 locus and TRB in K293_Cas9 cells. The indel rate at the C2 locus is shown.

[0287] [Table 8] JPEG2025138662000033.jpg254170JPEG2025138662000034.jpg203170

[0288] Example 5: Human CD3 + TRBC-guided screening of T cells The indel editing rate was high in the HEK293_Cas9 cells obtained in Example 4 TRBC guide, human CD3 + Editing efficiency and T cell receptor (TCR) in T cells Nucleofection, editing analysis, and TCR expression were performed. The analysis was performed as in Example 3. Table 9 and Figure 5 show the CD3 + Various guides for T cells Table 10 and Figure 6 show the results for indel frequency after TRBC editing by the nucleotide sequence. , CD3 + Measurement of TCR expression in T cells after TRBC editing (proportion of TCR-negative cells and (measured as a function of time) [Table 9] [Table 10]

[0289] Example 6: Off-target analysis of TRBC guide and TRAC guide Biochemical methods (e.g., Cameron et al., Nature Methods, Vol. 6, p. 600–606; see 2017) to identify TRAC, TRBC1, or TRBC Determine potential off-target genomic sites cleaved by Cas9 targeting 2 The guides that showed the most CD3-negative cells in Examples 3 and 5 were used in this experiment. Potential off-target genomic cleavage sites were investigated using a genomic DNA assay. Seven human TRAC-targeting sgRNAs, six TRBC-targeting sgRNAs, and off-target Two control guides with known genomic DNA sequences were isolated from the HEK293_Cas genome. The biochemical assay was performed using an RNP concentration of 16 nM. The number of potential off-target sites is plotted in Figure 7A and shown in Table 11. Potential off-target sites were identified for the tested sgRNAs.

[0290] [Table 11]

[0291] Targeted sequencing to validate potential off-target sites Known off-target detection assays, such as the biochemical methods used above, typically involve: In other situations, for example, a "broad search" is performed to find potential sites that can be tested in primary cells of interest. The "cast a wide net" design allows for a large number of potential For example, biochemical methods can be used to recover purified target sites free of the cellular environment. This is because it utilizes high molecular weight genomic DNA and depends on the dose of Cas9 RNP used. , usually account for a large proportion of the number of potential off-target sites. Potential off-target sites identified by Targeted sequencing of the site can be used to verify.

[0292] In one approach, Cas9 and the sgRNA of interest (e.g., for evaluation of potential A sgRNA (with a specific off-target site) is introduced into primary T cells. T cells are lysed and NG using primers flanking potential off-target sites. Amplicons are generated for S analysis. Identification of a certain level of indels is a potential While this may demonstrate off-target sites, it may also be a potential off-target site. The lack of indels found in the genomic DNA fragments may account for false positives in the off-target assays used. It may be indicative of.

[0293] This assay was used to identify guides showing on-target indel activity and identify potential off-target genes. The target genomic cleavage site was tested. In this experiment, human TRBC1 or human Five sgRNAs targeting TRBC2 and three targeting human TRAC were used. The guide was compared with a control guide, SEQ ID NO: 194 (VEGFA), which has known off-target properties. Both were performed using genomic DNA isolated from a pool of male human peripheral blood mononuclear cells (PBMCs). , and were screened. Potential detected using a guide concentration of 64 nM in the biochemical assay. The number of potential off-target sites is shown in Figure 7B and Table 12.

[0294] [Table 12]

[0295] Example 7: Use of TRAC and TRBC guides for multiplex editing The guides were also examined for simultaneous editing of the TRAC and TRBC loci. Peripheral blood mononuclear cells (PBMCs) from three different healthy donors were cultured in a lymphoprep ( Isolation from buffy coat using Stem Cell Technology (TM) and density gradient These cells were then centrifuged. CD3 / CD28 (Invitrogen) according to the manufacturer's instructions. T cells were activated and sorted. T cells were cultured in 5% FBS and IL7 and IL15 ( X-VIVO™ 15 Hematopoietic Serum-Free Culture Media (Lonza (trademark)) 106 Two days after stimulation, T cells were transfected with crRNA targeting TRAC. and TRBC-targeting crRNA simultaneously as described in Example 3. Cells from one donor were used to generate TRAC guides and T The RBC guides were electroporated in their isolated state. Six days after stimulation, The cells were cultured in 5% FBS and IL7 and IL15 (5 ng / ml each). 1 x 10 in X-VIVO™ 15 supplemented with 6 The cells were seeded at a concentration of 1 / ml. TRAC and TRBC knockout efficiencies were measured using flow cytometry, as shown in Figure 8A and Table 13. was assessed by assessing the percentage of T cells lacking CD3 molecules by cytometry. .

[0296] [Table 13]

[0297] Simultaneous editing of TRAC and TRBC using lentiviral insertion of TCR genes Three days after stimulation, the edited T cells were transfected with WT1-specific T cells derived from a healthy donor (HD1-TCR). Transduction was performed using a lentiviral vector (LV) encoding CR, and the LV was randomly transduced. Briefly, the TCR α and β chain genes were isolated, codon-optimized, and inserted into The HD1 TCR was cysteine ​​modified and cloned into LV under a bidirectional promoter. The DNA and amino acid sequences of the α-chain gene and β-chain gene are shown below. The β-chain was cloned in antisense orientation under the small human CMV promoter. The LV was cloned in the sense orientation under the promoter. Vesicular stomatitis virus (VSV) envelope was packaged using a third-generation construct. T lymphocytes were infected with this LV for 24 hours. 0 6 Two days after transduction, specific Vβ genes (TRBV 12-3 / TRBV12-4) and specific dextramer (epitope: VLDFAP The transduction efficiency was evaluated by determining the percentage of T cells expressing PGA. On day 15, T cell phenotype was assessed by cytofluorometry. Figure 8B and Table 14 show that over 45% The results showed that T cells expressed CD3 molecules (mean ± SEM = 45.3 ± 5.7). WT1-specific CD8 was assessed by measuring the dextramer-positive rate. + T The percentage of cells was over 95% (mean ± SEM = 95.3 ± 0.7). -WT1-specific CD8 assessed by measuring the positive rate + The proportion of T cells is When SEQ ID NO: 6 and SEQ ID NO: 95 were used, the results were greater than 95% (mean ± SEM = 95,3 ± 0, 7) (Figure 8C, Table 14). The T cell phenotype of the edited T cells was confirmed at day 15 as shown in Figure 8D and and were evaluated as shown in Table 15.

[0298] SEQ ID NO: 250: HD1 TCR alpha chain: DNA sequence ATGGAAACCCTGCTGAAGGTGCTGAGCGGCACACTGCTGT GGCAGCTGACATGGGTCCGATCTCAGCAGCCTGTGCAGTC TCCTCAGGCCGTGATTCTGAGAGAAGGCGAGGACGCCGTG ATCAACTGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACT GGTACAGACAGAAGCACGGCGAGGCCCCTGTGTTCCTGAT GATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAG ATCAGCGCCAGCTTCAACGAGAAGAAGCAGCAGTCCAGCC TGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGCACCTA CTTTTGTGGCACCGCCTGGATCAACGACTACAAGCTGTCT TTCGGAGCCGGCACCACAGTGACAGTGCGGGCCAATATTC AGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAA GAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGAC AGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGT ACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGA CTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGC GATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCC CCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGA CGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAAC CTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCC TGCTGCTCAAGGTGGCCGGCTTCAACCTGCTGATGACCCT GAGACTGTGGTCCAGCTGA

[0299] Accession No. 251: HD1 TCR α chain: Amino acid sequence METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAV INCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEK ISASFNEKKQQSSLYLTASQLSYSGTYFCGTAWINDYKLS FGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFD SQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0300] Accession No. 252: HD1 TCRβ chain: DNA sequence ATGGGATCTTGGACACTGTGTTGCGTGTCCCTGTGCATCC TGGTGGCCAAGCACACAGATGCCGGCGTGATCCAGTCTCC TAGACACGAAGTGACCGAGATGGGCCAAGAAGTGACCCTG CGCTGCAAGCCTATCAGCGGCCACGATTACCTGTTCTGGT ACAGACAGACCATGATGAGAGGCCTGGAACTGCTGATCTA CTTCAACAACAACGTGCCCATCGACGACAGCGGCATGCCC GAGGATAGATTCAGCGCCAAGATGCCCAACGCCAGCTTCA GCACCCTGAAGATCCAGCCTAGCGAGCCCAGAGATAGCGC CGTGTACTTCTGCGCCAGCAGAAAGACAGGCGGCTACAGC AATCAGCCCCAGCACTTTGGAGATGGCACCCGGCTGAGCA TCCTGGAAGATCTGAAGAACGTGTTCCCACCTGAGGTGGC CGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACAG AAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATCCCG ATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGT GCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGAAAGAG CAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCA GACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAA CCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAG AACGATGAGTGGACCCAGGATAGAGCCAAGCCTGTGACAC AGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGTGG CTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCC ACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGT ACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGT CAAGCGGAAGGATAGCAGGGGCTGA

[0301] SEQ ID NO: 253: HD1 TCR β-chain: Amino acid sequence MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTL RCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMP EDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRKTGGYS NQPQHFGDGTRLSILEDLKNVFPPEVAVFEPSEAEISHTQ KATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKE QPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSA TILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*

[0302]

Table 14

[0303]

Table 15

[0304] T cells with TRAC and TRBC editing and LV HD1-TCR insertion Dextramer was tested for its ability to kill primary AML blasts. ) WT1-CD8 staining and magnetic sorting + To enrich for specific T cells, Edited T cells were magnetically sorted. 21 days after transduction, WT1-CD8 + T thin The cells were cultured in primary A cells obtained from three different patients with the HLA-A*02:01 allele. These were co-cultured with ML blasts and, as controls, with primary blasts that did not possess the specific HLA alleles listed above. Co-cultures were performed at different effector-to-target ratios (1:1; 5:1; 10:1). After 6 hours of co-culture, the expression of caspase 3 in live target cells was measured as a readout. The results are shown in Figures 9A to 9D and Table 16. -Up to 43% of leukemic blasts with A*02:01 (mean ± SEM = 31 ± 6.6) Induction of apoptosis was observed in cells that do not express the HLA-A*02:01 allele. There was no detectable induction of apoptosis in AML blasts.

[0305] [Table 16]

[0306] Example 8: Additional TRAC and TRBC guides for multiplex editing In addition, additional crRNA was used for simultaneous editing of the TRAC and TRBC loci. T cells were treated with an RNP comprising SEQ ID NO: 185, an RNP comprising SEQ ID NO: 6, or The cells were subjected to nucleofection with a mixture of both RNP species. β-mercaptoethanol, 10 mM N-acetyl-L-cystine, 1x penicillin Streptomycin solution (Corning, Cat. No. 30-002-CI) was added and the IL7 (5 ng / ml), IL15 (5 ng / ml), and IL2 (200 ~500U / ml) was added to BioWhittaker™ X-VIVO™ )15 Hematopoietic Serum-Free Culture Med The same procedure as in Example 3 was carried out except that ia (Lonza (trademark), catalog number 04-418Q) was used. Seventeen days after nucleofection, T cells were harvested and analyzed by NGS. Figure 10 and Table 17 show the editing at each locus after co-nucleofection. Flow cytometry was used to detect surface T Editing efficiency can also be assessed by measuring the percentage of T cells expressing the CR protein. .

[0307] [Table 17]

[0308] Example 9 Additional TRAC- and TRBC-targeting crRNA pairs were synthesized as described in Example 7. Combinatorial editing was investigated. T cells were cultured with an RNP containing SEQ ID NO: 95, an RNP containing SEQ ID NO: 2, and an RNP containing SEQ ID NO: 3. TRAC was subjected to nucleofection with either RNP or a mixture of both RNP species. and TRBC knockout efficiency was measured by flow cytometry as shown in Figure 11A and Table 18. This was assessed by assessing the proportion of T cells lacking CD3 molecules by immunofluorescence.

[0309] [Table 18]

[0310] After lentiviral transfection, T cells Phenotype was assessed by cytofluorometry. WT1-specific CD8 + The percentage of T cells was measured using dextrama The T cell count of edited T cells was evaluated by measuring the positive value (Figure 11C, Table 19). The current model is shown in Figure 11D and Table 20.

[0311] [Table 19]

[0312] [Table 20]

[0313] In the modified cells of Examples 7 and 9, the TCR α chain of SEQ ID NO: 251 and the TCR α chain of SEQ ID NO: 2 Lentiviral vector encoding WT1-specific TCR with 53 TCR β chains The TRAC gRNA and TRBC gRNA are combined in the above-mentioned modified form. The transformed cells can be modified by delivering a first composition and a second composition to the cells; The first composition comprises: (a) a guide RNA comprising a sequence selected from the following: (i) SEQ ID NO: (ii) a guide sequence selected from SEQ ID NOs: 1 to 89; At least 17, at least 18, at least 19, or at least 20 consecutive (iii) at least 99 nucleotides for a sequence selected from SEQ ID NOs: 1 to 89 %, at least 98%, at least 97%, at least 96%, at least 95%, at least at least 94%, at least 93%, at least 92%, at least 91%, or less (iv) a guide sequence having 90% identity with any one of SEQ ID NOs: 1 to 24; and (iv) a guide sequence comprising any one of SEQ ID NOs: 1 to 6. or (b) a nucleic acid encoding the guide RNA of (a.); and, optionally, (c) an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent said second composition comprising a TCR α chain of SEQ ID NO: 251 and a TCR β chain of SEQ ID NO: 253; The lentiviral vector encoding a WT1-specific TCR having the WT1-specific TCR chain and the WT1-specific TCR chain is included.

[0314] In some embodiments, the modified cells deliver a composition further comprising: (a) a guide RNA comprising a sequence selected from the following: ) SEQ ID NO: 90 to 178, SEQ ID NO: 185, and SEQ ID NO: 213 to 218 (ii) guide sequences of SEQ ID NOs: 90 to 178, 185, and 213; At least 17, at least 18, at least 19 of the sequences selected from ∼218 or at least 20 consecutive nucleotides; (iii) SEQ ID NOs: 90-178, No. 185, and at least 99 for a sequence selected from SEQ ID NOs: 213 to 218 %, at least 98%, at least 97%, at least 96%, at least 95%, at least at least 94%, at least 93%, at least 92%, at least 91%, or less (iv) a guide sequence having 90% identity with SEQ ID NOs: 90 to 113 and SEQ ID NO: 213 to 218; and (iv) a guide sequence comprising any one of SEQ ID NO: 90. or (b) a guide sequence comprising any one of the following: A nucleic acid encoding

[0315] Example 10. Single-guide editing The following examples were carried out according to the following methods unless otherwise specified in each example.

[0316] Genomic DNA isolation T cells were harvested >48 hours after nucleotransfection. Isolation was carried out as described in Example 1. DNA samples were PCR purified as described in Example 1. After that, NGS analysis was performed.

[0317] Delivery of RNP to T cells Purchase healthy donor PBMCs or Leukopak and follow the manufacturer's protocol , StraightFrom CD4 / CD8 Microbead Kit (Miltenyi By positive selection using EasySep Human T-cell Isolation Kit (Stem Cell Technology) T cells were isolated by negative selection using a IgG antibody (Cat. No. 17951) T cells were frozen in Cryostor CS10 freezing medium (Catalyzed) for further use. After thawing, T cells were cryopreserved in 200 U / mL IL2 (log no. 07930). Peprotech), and 5 ng / mL IL7 and IL15-supplemented basal medium (2-mercaptoethanol, N-acetyl-cysteine, The cells were then left overnight in XVIVO15 supplemented with 5% human AB serum or FBS. After that, the T cells were transfected with T Cell TransAct (Miltenyi Biotec, 130- 111-160) as recommended by the manufacturer and incubated for 48-72 hours. Then, electroporation was performed.

[0318] The ribonucleoprotein (RNP) complex containing the Cas9 protein and ggRNA is First, separate the individual samples by mixing the respective volumes of reagents, incubating at 95°C for 2 minutes, and cooling to room temperature. The guide RNA was prepared by preannealing the crRNA and trRNA. As a single RNA molecule (single guide RNA, sgRNA), Alternatively, pre-animate by incubating at 95°C for 2 minutes followed by cooling to ambient temperature. The two separate RNA molecules (dual guide RNA, dgRNA) rRNA and trRNA were used. SEQ ID NO: 186 (TRAC) or SEQ ID NO: 180 (TRBC1 / 2) was mixed with SpyCas9 at a molar ratio of 2:1 to generate TR RNPs targeting the AC locus or the TRBC locus were complexed to a final concentration of 20 For electroporation, T cells were incubated for 5-20 min at 4°C. x10 6 TRAC RNP and TRBC RNP were suspended in cells / 100 μL. T cells were treated with P3 Primary C ell 96-well Nucleofector™ Kit (Lonza, Catalog) Product code V4SP-3960) or Lonza 4D-Nucleofector XU nit (Cat. No.: AAF-1002X) and Buffer P3. The cells were subjected to nucleofection with RNP using the manufacturer's pulse code. Immediately after poration, basal medium was added to the cells and the cells were allowed to rest for at least 4 hours.

[0319] For sorting assays, cells were grown using the G-Rex protocol. After electroporation, 1 × 10 6 100 cells per well of a 24-well G-Rex Transfer 1 ml of cytokine medium into the well and add 10 cells at an MOI of 3 x 10. 5 virus equivalent to After 24 hours, the volume was adjusted to 7 ml with cytokine medium. Every 2-3 days, half of the medium (3.5 ml) was removed without disturbing the cells, and 2× Cytokine medium was added, and the cells were harvested on days 11-13.

[0320] Cell staining for flow cytometry Four to 12 days after RNP nucleofection, the edited T cells (200 μl / 200,0 00 cells) were collected into a 96-well round-bottom plate and spun down at 500 g for 5 minutes. , cells, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD45RO antibody, anti-CD4 5RA antibody, anti-CD27 antibody, anti-CCR7 antibody, anti-CD62L antibody, and / or TC Antibody mixtures containing combinations of TCR-specific pMHC tetramers or TCR-specific Vβ chain antibodies The mixture was incubated at room temperature in the dark for 45 minutes. Spin down at 0g for 5 min, collect the cell pellet, and add 1:10,000 DAPI (by The cells were suspended in FACS buffer (Oregon, #422801). Flow cytometry was performed using lex according to the manufacturer's instructions.

[0321] Single guides were transduced into TRAC and TRBC loci of stimulated T cells. Individual and simultaneous editing was also tested. T cells were transfected with the TRBC1 locus and TR RNP containing guide SEQ ID NO: 180, which targets the BC2 locus, targets TRAC RNP containing the guide SEQ ID NO: 186, or a 2:1 ratio of guide to Cas9. Two days after electroporation, the cells were subjected to nucleofection with a mixture of NP species. , T cells were passaged, and lysates were collected.

[0322] Genomic DNA was prepared and NGS analysis was performed as described in Example 1. Samples were tested. Triplicates were performed and a single mock sample was used. The knockdown efficiency of TCR cell surface expression for individual and simultaneous editing of the CD4+ TCRs was evaluated using CD4+ TCR knockdown assays. The presence or absence of CD3 was assessed by targeted flow cytometry. The percentage of deleted T cells is shown in Table 21 and Figure 12A.

[0323] [Table 21]

[0324] CD3 - After individual and combined editing of TRAC and TRBC in T cells Indel frequencies were assessed by NGS analysis of the TRAC and TRBC. The percentage of edited sequences is shown in Table 22 and Figure 12B.

[0325] [Table 22]

[0326] Example 11. TRAC guide insertion screening Insertion of AAV templates into T cells Using 12 guide positions targeting the TRAC locus, 8 of the TRAC locus were By removing the 0 bp region, cleavage at that site is prevented after repair, and the guide / Cas9 is able to use the template. We designed an adeno-associated virus (AAV) template that prevents cleavage from the nucleotide sequence. This creates a locus that allows the insertion of a TCR into the TRAC locus. Using the homology arms, insert a model TCR and insert GFP into the second one. Thus, we designed a construct in which both genes were driven by the EF1a promoter. The BglI was synthesized by GenScript USA Inc. I restriction enzyme recognition site into pUC19 vector and subcloned into AAV vector The homology arms were placed adjacent to the TRAC-guided cleavage site, and the TCR was then cloned. or a gene encoding a reporter molecule (e.g., GFP) into T cells in a site-specific manner. An AAV template was designed to

[0327] T cell isolation and RNP nucleotransfection procedures were performed as described in Example 10. Ten minutes after electroporation, 1x10 T cells were 6 Cytokine in cells / mL Add 3x10 AAV template to the incubation medium. 5 Electroporation After 24 hours, cells were split and cultured for an additional 4–12 days in static culture or in G-REX media. Rutiwell Plate (Wilson Wolff Manufacturing Co.) and then used for functional analysis. Flow cytometry was performed.

[0328] TRAC guide insertion screening Dual guides targeting the first exon of the TRAC locus were used to screen T cells. In the first screening, the GFP reporter gene was inserted into the chromosome and the insertion efficiency was evaluated. An AAV-based insertion template (AV9) encoding the vector was used. The AV9 design included 8 A 0 bp gap was designed to encompass the target sites of the guides listed in Table 23. V9 is listed below in Table 37. AAV only samples and RNP were used as controls. Except for the mock sample, duplicates were run using RNPs containing dual guides. T cells were nucleofected with the guide-to-Cas9 ratio set at 1:1. performed the cell transduction procedure using RNP as described in Example 10. The insertion efficiency of the C-guide was determined by flow cytometry as described in Example 10. GFP fluorescence from the inserted construct was detected as shown in Table 23 and Figure 13A. Efficiency of knockdown of TCR cell surface expression with TRAC-guided editing was assessed by flow cytometry targeting the presence or absence of CD3. The percentage of T cells lacking the three molecules is shown in Table 23 and Figure 13B.

[0329] [Table 23]

[0330] Example 12. Single guide screening for TRAC insertion Next, a single guide targeting the first exon of the TRAC locus was engineered to express GATA in T cells. The insertion efficiency using AV9 was evaluated by screening using a guide-to-Cas9 ratio of 1:1. T cell generation and immunization were performed in duplicate as described in Example 10, except that The RNP transduction procedure was performed using the guides listed in Table 24. Designed as described in Example 11. NGS indel analysis was performed as described in Example 1.

[0331] The insertion efficiency of the TRAC guide was determined by flow cytometry, as shown in Table 24 and Figure 2 GFP fluorescence from the inserted construct was detected as shown in 14A. The efficiency of knockdown of TCR cell surface expression by C-guide was evaluated in the presence or absence of CD3. The percentage of CD3-deficient T cells was assessed by targeted flow cytometry. The editing efficiency of chemically modified and unmodified guides was also assayed as shown in Table 24 and Figure 14B. It was found to have activity.

[0332] [Table 24]

[0333] Example 13: Evaluation of promoters and ITRs for insertion and expression of exogenous TCRs Engineering gene-edited TCR-T cells for functional analysis TCR insertion and subsequent cell surface expression were confirmed using various promoters and two ITR lengths. T cell isolation and RNP transfection using guide SEQ ID NO: 185 The procedure was performed as in Example 10, except that a 1:1 molar ratio of gRNA to SpyCas9 was used. RNP transduction of TCR-C-containing AAV constructs into T cells was performed as described. Transduction was carried out as described in Example 11. The AAV template contained a sequence within the TRAC locus. 500 bp homology corresponding to the 500 bp flanking the 185 cleavage site Each AAV construct contained a promoter, as shown in Table 25. The edited T cells were characterized by different combinations of target and ITRs. Tetramer staining for flow cytometry analysis was performed as described above.

[0334] The efficiency of TCR-C insertion was assessed by cytofluorimetric analysis, and the presence of CD3+ on the cell surface was assessed. T cell identity is determined by the TCR's specific ligand, the tetramer RMF peptide. Both the number of cells expressing the inserted TCR and the number of cells expressing the inserted TCR were assessed (e.g., See U.S. Patent Application Publication No. 20160083449, which is incorporated herein by reference. The percentage of CD3+ tetramer+ cells is shown in Table 25 and Figure 15A. The mean fluorescence intensity (MFI) is shown in Table 25 and Figure 15B.

[0335] [Table 25] The sequence elements and sequences shown in this table are further defined in Table 37.

[0336] Example 14: GFP expression in engineered T cells using the endogenous TRAC promoter In a separate experiment, a GFP reporter gene was included after the P2A ribosomal skipping site. An AAV template (AV10) (e.g., without an exogenous promoter) was inserted into the first nucleotide of the TRAC locus. 1 exon and whether the endogenous TRAC promoter can drive GFP expression. As described in Example 10, RNP containing the guide SEQ ID NO: 186 was transfected into T cells. Nucleofection was performed using 100% ribosomal RNA (RRNA) and transduction was performed using AAV AV10. As a control, unedited T cells (mock) and those that underwent RNP nucleofection but not AAV were used. After 4 days of culture, the CD3 T cells of the edited T cells were were co-stained with APC-Cy7-conjugated anti-CD3e (Biolegend, 300318). Flow cytometry analysis of GFP expression and TCR knockout revealed: The results are as shown in Table 26 and Figures 16A to 16C. [Table 26]

[0337] Example 15: Modification using endogenous and exogenous TRAC promoters TCR expression on mutant T cells In additional experiments, TCR-A, TCR-C, with or without an exogenous promoter were expressed. An AAV template containing TCR-D or TCR-D is inserted into the TRAC locus to inhibit the endogenous TRAC gene. We then confirmed whether the promoter could drive the expression and function of the inserted TCR. Cell transfection and AAV insertion are described in Examples 10 and 11, respectively. T cells were transfected with TRAC (SEQ ID NO: 186) and Nucleofection was performed using RNP containing TRBC (guide sequence number 180). The guide-containing RNPs contained various AAV templates as shown in Table 27. TCR-expressing T cells were identified in CD4+ and CD8+ cells by the inclusion of TC The results were detected by flow cytometry using an antibody specific for the TCRV β chain of R. Table 2 7 and 17A to 17C (in the case of TCR-A, anti-Vβ8, [Bio Legend catalog number 140104]; Vβ7.2 for TCR-D, Figure 17B [ Beckman Coulter, Inc., catalog number IM3604]; Vβ17 for TCR-C , illustrated in Figure 17C [Beckman Coulter, Catalog No. IM2048]).

[0338] [Table 27] The sequence elements and sequences shown in this table are further defined in Table 37.

[0339] Degranulation and cytokine release of T cells in response to specific WT1 peptide antigens were investigated. , modified effector expressed from the endogenous promoter or the exogenous TRAC promoter. Specifically, the engineered T cells were then injected into the cancer cell line OCI-AML. After co-culture with peptide-pulsed cells derived from 3 (DSMZ, Cat. No. ACC582) The increased expression of CD107a, as well as the expression of IL2 and IFN-γ were evaluated. Briefly, 100,000 OCI-AML3 cells were cultured in basal medium (Xvivo basal medium) for 12 days. Medium: No cytokines + 1 μL / mL GolgiPlug + 0.7 μL / mL Go lgiStop + 30 μL / mL CD107a APC / Cy7 (3 μL / well) Increasing amounts of the 9-mer peptide VLDFAPPGA(VLD) or The peptide concentrations ranged from 0 to 5000 nM. Gene-edited TCR+ T cells were administered at a dose of 1×10 6 After TCR insertion, the number of cells / ml The final concentration was 30 μl / ml CD107a APC / Cyanine7 antibody ( Olegend), 1 μl / ml Golgiplug (BD), and 0.7 μl / m l Suspend the cells in XVIVO15 basal medium containing Golgistop (BD) and The target cells were added to the peptide-pulsed target cells at an E:T ratio of 1:1. The co-cultures were incubated overnight at 37°C and the cells were analyzed for surface markers CD3, CD4, and CD6. D8, and stain specific TCR β chains for TCR-A or TCR-D, for 30 min. After surface staining, the cells were fixed and incubated using a commercially available kit (Invitrogen). The cells were then permeabilized and stained for intracellular IFN-β and TNFα. After intracellular staining, cells were washed and analyzed by flow cytometry. Ta.

[0340] EF1a (or EF-1α) and endogenous factors for T cell killing and cytokine release The effect of endogenous promoters was assessed by the presence of CD107a, IFNγ, and / or TCR-bearing effectors were measured by the percentage of cells positive for TNFα or TNFα. The TCR+ T cell and promoter types are listed in Table 28. Cytometry results are shown in Table 28, and the dose response for TCR-A expressing cells. The curves are shown in Figure 18A (AV21 and AV11). CD107a expressing TCR-D The percentage of + cells is shown in Table 28 and Figure 18B (AV20 and AV18). The immune response induced by the IL-1 receptor was assessed by measuring the proportion of TNF-α and interferon-γ. The evaluation results are shown in Table 28 and Figures 19A and 19B. [Table 28] TIFF2025138662000055.tif216168

[0341] Example 16: TCR expression by knockout of both the endogenous T cell receptor α and β chains Increased T cells were treated with a modified TCR-A containing both an inserted α chain and an inserted β chain, Cell surface expression of TCR-B or TCR-D was assessed. Three conditions were tested to increase β-strand pairing: (1) forming cysteine ​​bridges; (2) the addition of a cysteine ​​residue that can link the inserted α and β chains; C promoter or from an exogenous promoter, and (3) TRAC Disruption of endogenous copies of TRBC1, TRBC2, and TRBC3. T cell transfection and and AAV transduction was performed as described in Example 10. TCR was expressed as described above. RNPs targeting only the TRAC locus, or the TRAC locus and TRBC1 gene The AAV template, AV, contains RNPs targeting both the TRBC2 locus and the TRBC3 locus. Transfection was performed via AV11, AV13, AV18, or AV20. Afterwards, the T cells were expanded as described in Example 10. After 9 days of cell expansion, the edited T cells were , co-stained with anti-CD3e and the appropriate V-β reagent (PE), and analyzed via flow cytometry. The modified TCRs were identified by the following: TCR-A and TCR-B CD3+Vb The percentage of 7.2+ cells is shown in Table 29. Figures 20A and 20B show the data in Table 29. The cell surface expression of modified TCR-A and TCR-B CD3+Vb7.2+ cells 1 shows representative plots of modified T cells in CD8+ and CD4+ cell populations. Cell surface expression of TCR-A and modified TCR-B was also measured, as shown in Table 29 and Figure 21C The result was as shown in the figure.

[0342] [Table 29]

[0343] AV20-TCR-D and AV20-TCR-D vectors using endogenous or exogenous promoters The percentage of AV18-TCR-D CD3+Vb7.2+ cells is shown in Table 30. We measured cell surface expression of modified TCR-D in the CD4+ and CD4+ cell populations. The results are as shown in Table 30 and Figures 21A to 21D.

[0344] [Table 30]

[0345] Example 17: Effect of TRBC knockout on pairing of α and β chains from additional TCRs influence T cells were subjected to the influence of the endogenous β chain on pairing of both the inserted α chain and the inserted β chain. The effect of MF-1 on TCR expression was assessed using additional modified TCRs. The AAV inserts in Table 31 were used for transduction, as described herein, except that I was measured and the AAV inserts in Table 31 were used for transduction. After 9 days of cell expansion, modified T cells were co-stained with VLD-tetramer ( Orthogonal V-β staining method), cells with TCR cell surface expression were analyzed by flow cytometry The results are shown in Table 31. The data obtained from each experiment are shown in Figure 22 and Figure 33. 23 and 24. In addition, mean fluorescence intensity (MFI) was calculated by flow cytometry. The results are shown in Table 31 and Figures 25 and 26.

[0346] [Table 31]

[0347] Example 18: Toxicity and cytokine release of modified T cells The engineered T cells were assayed for cytotoxicity and cytokine responses in target cells, respectively. Upregulation was achieved through measurement of apoptosis and cytokine release into the growth medium. RNPs that target only the TRAC locus or both the TRAC locus and TRB Using the AAV template sequences listed in Table 32, which have RNPs targeting both the C locus T cells were engineered as described herein. The TCR+ T cell ratio was 2.5:1 The HLA-02.01-positive T2 cell line target was assayed as described in Example 15, except that E:T was used. Cells were pulsed and co-cultured with engineered T cells. Caspase 3 / 7Red reagent (Essence Biosciences, Inc.) was used. A final concentration of 2 μM of Essen Bioscience was added to each well. The Incucyte Live Cell Analysis System m (Essen Biosciences) and Incucyte S3 analysis software ( Using the 2018B version, caspase 3 / 7 activity was measured from apoptotic cells after 6 hours. After 24 hours, the supernatant was collected from each well and analyzed for IL2 and IFN-γ. Cytokine release was measured using Duoset ELISA kit (R&D Systems, Inc.) manufactured by The mean caspase 3 / 7 integrated intensity was quantified by ELISA using the protocol of the authors. are shown in Tables 32 and 33, and in FIGS. 27A to 27C and 28A to 28D. Cytokine release is shown in Table 34AD and in Figures 29A-29G and 30A-30D. Shown below.

[0348] [Table 32] [Table 33] TIFF2025138662000061.tif43160 [Table 34-1] [Table 34-2] [Table 34-3] TIFF2025138662000065.tif41160 [Table 34-4] TIFF2025138662000067.tif107163

[0349] Example 19: Toxicity of modified T cells: CD107a degranulation and intracellular cytokine staining ( Quantitation by ICS The engineered T cells were also assayed for cytotoxicity and cytokine responses using CD107 The TRAC gene locus was targeted and quantified through measurement of a and intracellular cytokine staining. RNPs that target the TRAC locus or both the TRBC locus T cells were transfected as described in Example 18 using the AAV template sequences listed in Table 35, each having the following structure: The edited TCR+ T cells were then injected into peptide-pulsed target cells at a 1:1 E:T cell ratio. HLA-02.01-positive T2 cell lines were pulsed as described in Example 18, except that The co-cultures were incubated overnight at 37°C and the cells were incubated for 10 min to detect the surface markers CD3, CD4, and , CD8, and specific TCR β chains or tetramers. After surface staining, cells were fixed. The cells were then permeabilized and intracellular IFN-γ and IFN-γ were detected using a commercially available kit (Invitrogen). The immune responses elicited against TCR expression were shown in Table 35A and As shown in Figures 31A-31B, cells resulting from TCR expression with TRBC knockout Toxicity was assessed by measuring CD107a in the engineered T cells, as shown in Table 3. 5B and 32A to 32B. [Table 35-1] TIFF2025138662000069.tif60149 [Table 35-2] TIFF2025138662000071.tif56160

[0350] Example 20: Alloreactivity Assay The non-specific reactivity of the modified T cells prepared in Example 18 to antigen-presenting cells was investigated. The modified T cells were then quantified using a mixed lymphocyte reaction (MLR) suppression assay. CellTrace Violet (Invitrogen) was used according to the manufacturer's instructions. CD3+ cells were depleted from allogeneic PBMCs using MACS (Miltenyi). Specifically, 5 × 10 4 3 x 10 T cells 4 The cells were seeded in a 96-well U-bottom plate (Corning) along with CD3-depleted allogeneic PBMCs. After 5 days, cells were harvested, stained with anti-CD4, and analyzed by flow cytometry. The extent of proliferation was quantified by dilution of CellTrace Violet dye. Roswell Park Memorial Institute (RPMI) containing glutamine Recombinant Recombinant Microbial Incubation Medium (RPMI; Corning) 1640 medium (RPMI; Corning) containing 10% Fe S, 1x GlutaMAX, 10mM HEPES, 1x penicillin / streptomycin Supplemented with 1 mM sodium pyruvate, 50 μM 2-ME, and 1× essential amino acids. This was used throughout the MLR assay. The average percentage of stained cells with CTV (Cell Violet), i.e., cells with high proliferation values, was calculated as follows: The results are shown in Table 36 and Figures 33A and 33B. Figures 33A and 33B show the CTV level The gating used for the assay is shown in Table 36 and Figures 33C and 33D. As shown, when each inserted TCR was tested, CD4+ cells and CD4- (CD In both TRBC RNP-treated and TRBC RNP-treated cells, the TRBC RNP-treated cells were significantly more potent than the TRBC RNP-treated cells. showed less proliferation than untreated cells.

[0351] [Table 36]

[0352] All templates were delivered as ssAAV and contained the 5' and 3' ITRs of AAV2. Note that the homology arms are as above. Many additionally have the following: As shown, the sequence may contain one or more promoters, truncations, or polyA sequences. indicates a stop codon. [Table 37] TIFF2025138662000074.tif14118

Claims

1. A method for modifying the DNA sequence within the TRBC1 gene and / or the TRBC2 gene. So, delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent acid, A method comprising:

2. 1. A method for modifying a DNA sequence, comprising: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from: i. Table 1 and / or for SEQ ID NOs: 1-89 and 179-184 is 15 consecutive nucleotides ±10 of the genome coordinates listed in any of Table 3 a sequence comprising nucleotides; ii. at least 17, at least 18, at least 19 of the sequences from (i); or at least 20 consecutive nucleotides; iii. at least 99%, at least 98% to a sequence selected from (i); At least 97%, at least 96%, at least 95%, at least 94%, at least or at least 93%, at least 92%, at least 91%, or at least 90% identity. a guide sequence; (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent acid, A method comprising:

3. A method for reducing expression of the TRBC1 gene and / or the TRBC2 gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or b. a nucleic acid encoding the guide RNA of (a); and, optionally, c. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A method comprising:

4. 1. A method of immunotherapy comprising: administering the composition to the subject, their autologous cells, and / or allogeneic cells; The composition comprises: a. A guide RNA comprising a sequence selected from: i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or b. a nucleic acid encoding the guide RNA of (a); and, optionally, c. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A method comprising:

5. 1. A method for modifying a DNA sequence within a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from: i. a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218 the selected guide sequence; ii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a); and, optionally, c. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A method comprising:

6. 1. A method for modifying a DNA sequence, comprising: delivering the composition to the cell; The composition comprises: (a) a guide RNA comprising a sequence selected from: i. a sequence as set forth in any of Tables 2 and / or 3 for SEQ ID NOs: 90-218 A sequence containing 15 consecutive nucleotides ± 10 nucleotides of the genome coordinate ； ii. at least 17, at least 18, at least 19 of the sequences from (i); or at least 20 consecutive nucleotides; iii. at least 99%, at least 98% to a sequence selected from (i); At least 97%, at least 96%, at least 95%, at least 94%, at least or at least 93%, at least 92%, at least 91%, or at least 90% identity. a guide sequence; (b) a nucleic acid encoding the guide RNA of (a); and, optionally, (c) an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent acid, A method comprising:

7. 1. A method for reducing expression of a TRAC gene, comprising: delivering the composition to the cell; The composition comprises: a. A guide RNA comprising a sequence selected from: i. a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218 the selected guide sequence; ii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a); and, optionally, c. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A method comprising:

8. 1. A method of immunotherapy comprising: administering the composition to the subject, their autologous cells, and / or allogeneic cells; The composition comprises: a. A guide RNA comprising a sequence selected from: i. a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218 the selected guide sequence; ii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or b. a nucleic acid encoding the guide RNA of (a.); and, optionally, c. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A method comprising:

9. DNA sequences within the TRBC1 gene, TRBC2 gene and / or TRAC gene A method of modifying, A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and a nucleic acid encoding an RNA-guided DNA-binding agent or an RNA-guided DNA-binding agent. 、 The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; and a sequence selected from The second guide RNA is vi. SEQ ID NOs: 90-178, 185, and 213-218 the guide sequence selected; vii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or at least 20 of the sequences selected from at least 20 consecutive nucleotides; viii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, for a sequence selected from At least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence having at least 92%, at least 91%, or at least 90% identity; ix. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: x. a guide sequence comprising any one of SEQ ID NOs: 90-95; The method of claim 1, comprising a sequence selected from

10. Reduces expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene A method of A cell is administered a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA. and a nucleic acid encoding an RNA-guided DNA-binding agent or an RNA-guided DNA-binding agent. 、 The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; and a sequence selected from The second guide RNA is i. a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218 the selected guide sequence; ii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; iii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; iv. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: v. a guide sequence comprising any one of SEQ ID NOs: 90-95; The method of claim 1, comprising a sequence selected from

11. 1. A method of immunotherapy comprising: administering the composition to a subject, to autologous cells, or to allogeneic cells; The composition comprises a first guide RNA, a second guide RNA, and optionally an RNA guide and a nucleic acid encoding a DNA-binding agent or an RNA-guided DNA-binding agent, The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and v. a guide sequence comprising any one of SEQ ID NOs: 1-6; and a sequence selected from The second guide RNA is vi. SEQ ID NOs: 90-178, 185, and 213-218 the guide sequence selected; vii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or at least 20 of the sequences selected from at least 20 consecutive nucleotides; viii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, for a sequence selected from At least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence having at least 92%, at least 91%, or at least 90% identity; ix. SEQ ID NOs: 90-113, 185, and 213-218 a guide sequence comprising any one of the following: x. a guide sequence comprising any one of SEQ ID NOs: 90-95; The method of claim 1, comprising a sequence selected from

12. Methods for expressing heterologous immune receptors via intralocus insertion at the TRAC locus - Patent Application 20070122997 There was, A cell is administered a first guide RNA, a second guide RNA, and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA-binding agent; and The first guide RNA i. a guide sequence selected from SEQ ID NOs: 1-89; ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% guide sequences with identity; iv. a guide sequence comprising any one of SEQ ID NOs: 1, 2, 3, 5, 6; and v. a guide sequence comprising any one of SEQ ID NOs: 2, 3, 5, 6; and a sequence selected from The second guide RNA is vi. A moiety selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218 Id sequence; vii. selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218 At least 17, at least 18, at least 19, or at least 20 consecutive nucleotides; viii. A sequence selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218 At least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92% %, at least 91%, or at least 90% identity to a guide sequence; ix. A vector comprising any one of SEQ ID NOs: 90, 95, 97, 185, and 214. Guide sequence; x. a guide sequence comprising any one of SEQ ID NOs: 90, 95, and 185; and and xi. a guide sequence comprising SEQ ID NO: 90 or 214; The method of claim 1, comprising a sequence selected from

13. The first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 9 13. The method of claim 9, comprising an array of 0.

14. The first guide RNA comprises the sequence of SEQ ID NO: 180, and the second guide RNA comprises the sequence of SEQ ID NO:

13. The method of any one of claims 9 to 12, comprising the sequence of No.

186.

15. The first guide RNA comprises any one of the sequences of SEQ ID NOs: 1, 2, 3, 5, and 6.

13. The method of claim 9, wherein the second guide RNA comprises the sequence of SEQ ID NO:

90. The method described.

16. The first guide RNA comprises any one of the sequences of SEQ ID NOs: 1, 2, 3, 5, and 6.

13. The method of claim 9, wherein the second guide RNA comprises the sequence of SEQ ID NO:

214. The method described below.

17. the first guide RNA, the second guide RNA, and the RNA-guided DNA binding agent; or The nucleic acid encoding the RNA-guided DNA binder is administered substantially simultaneously.

17. The method according to any one of claims 16 to 16.

18. DNA sequences within the TRBC1 gene, TRBC2 gene and / or TRAC gene The method according to any one of claims 9 to 17, wherein the method is simultaneously modified.

19. The RNA-guided DNA-binding agent or a nucleic acid encoding the RNA-guided DNA-binding agent is introduced. The method according to any one of claims 1 to 18, wherein the

20. a. The TRBC1 gene, TRBC2 gene and / or genes in cells and / or subjects or introducing a double-strand break (DSB) in the TRAC gene; or b. TRBC1 gene, TRBC2 gene and / or genes in cells and / or subjects or introducing a single-strand break (SSB) within the TRAC gene; or c. The TRBC1 gene, TRBC2 gene and / or genes in cells and / or subjects or reducing the expression of the TRAC gene; The method of any one of claims 1 to 19, further comprising:

21. and optionally further comprising introducing a nucleic acid sequence encoding a polypeptide of interest. a. the one or more polypeptides of interest comprise a receptor; b. the one or more polypeptides of interest comprise an immune receptor; c. the one or more polypeptides of interest comprise a T cell receptor, and optionally: the T cell receptor recognizes a cancer antigen; d. the one or more polypeptides of interest comprise a WT1-specific T cell receptor; T cell receptor recognizes WT1 or a fragment thereof; e. the one or more polypeptides of interest comprise a chimeric antigen receptor, and optionally wherein the chimeric antigen receptor recognizes a cancer antigen; or f. the one or more polypeptides of interest comprise a WT1-specific chimeric antigen receptor; The chimeric antigen receptor recognizes WT1 or a fragment thereof. The method according to any one of claims 1 to 20.

22. a. Introducing the TCR alpha and TCR beta chains; b. Introducing one or more nucleic acid sequences encoding the TCR alpha and TCR beta chains ； c. Introducing the WT1-specific TCR α and β chains; d. Derivation of one or more nucleic acid sequences encoding the α and β chains of a WT1-specific TCR. Entering; e. Introducing a first TCR sequence selected from: (i) SEQ ID NO: 501, or SEQ ID NO:504; (ii) at least 99% to SEQ ID NO:501 or SEQ ID NO:504 %, at least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and (iii) at least 20, at least 30 of SEQ ID NO: 501 or SEQ ID NO: 504 , at least 40, at least 50, at least 60, at least 70, At least 80, at least 90, at least 100, at least 150, at least a contiguous subsequence of at least 200 or at least 250 amino acids, and, introducing a second TCR sequence selected from: (i) SEQ ID NO: 502 or sequence No. 505; (ii) at least 99% to SEQ ID NO: 502 or SEQ ID NO: 505; At least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and iii) at least 20, at least 30 of SEQ ID NO: 502 or SEQ ID NO: 505; At least 40, at least 50, at least 60, at least 70, at least At least 80, at least 90, at least 100, at least 150, at least 2 a contiguous subsequence of at least 200, at least 250, or at least 300 amino acids 、 f. Introducing a first TCR sequence selected from: (i) SEQ ID NO: 501; or SEQ ID NO:513; (ii) at least 99% to SEQ ID NO:510 or SEQ ID NO:513 %, at least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and (iii) at least 20, at least 30 of SEQ ID NO: 510 or SEQ ID NO: 513 , at least 40, at least 50, at least 60, at least 70, At least 80, at least 90, at least 100, at least 150, at least a contiguous subsequence of at least 200 or at least 250 amino acids, and, introducing a second TCR sequence selected from: (i) SEQ ID NO: 511 or sequence No. 514; (ii) at least 99% to SEQ ID NO: 511 or SEQ ID NO: 514; At least 95%, at least 90%, at least 85%, at least 80%, at least an amino acid sequence having at least 70%, or (of) at least 60% identity; and iii) at least 20, at least 30 of SEQ ID NO: 511 or SEQ ID NO: 514; At least 40, at least 50, at least 60, at least 70, at least At least 80, at least 90, at least 100, at least 150, at least 2 a contiguous subsequence of at least 200, at least 250, or at least 300 amino acids 、 g. Introducing a nucleic acid sequence comprising a sequence encoding the first TCR sequence of (e) or (f). thing; h. Introducing a nucleic acid sequence comprising a sequence encoding the second TCR sequence of (e) or (f). thing; i. introducing a nucleic acid sequence comprising the nucleic acid sequences of (g) and (h); j. SEQ ID NO: 500, 503, 506, 509, 512, 515, 518, or 5 21, or at least 99%, at least 9 5%, or at least 90% identity to the amino acid sequence of the target gene; by introducing a nucleic acid sequence encoding the same; k. A TCRα chain polypeptide and a TCRβ chain polypeptide selected from the following (i) to (vii): or at least 99%, at least 95%, at least Introduce an amino acid sequence with at least 90% identity: i) SEQ ID NO: 501 and SEQ ID NO: 502; ii) SEQ ID NO: 504 and SEQ ID NO: 505; iii) SEQ ID NO: 507 and SEQ ID NO: 508; iv) SEQ ID NO: 510 and SEQ ID NO: 511; v) SEQ ID NO: 513 and SEQ ID NO: 514; vi) SEQ ID NO: 516 and SEQ ID NO: 517; vii) SEQ ID NO: 519 and SEQ ID NO: 520; l. Nucleic acids encoding the TCR α and TCR β chain polypeptides of (k). Introducing an array, The method of any one of claims 1 to 20, further comprising:

23. 23. The method of claim 22, wherein the first nucleic acid sequence is flanked by sequences homologous to a first target locus. The method described below.

24. 23. The method of claim 22, wherein the second nucleic acid sequence is flanked by sequences homologous to a second target locus. The method described below.

25. the first target locus is the TRAC gene, the TRBC1 gene, or the TRBC2 gene The method according to any one of claims 23 to 24, wherein the gene is, for example, a TRAC gene.

26. the second target locus is the TRAC gene, the TRBC1 gene, or the TRBC2 gene and is, for example, the TRBC1 gene or the TRBC2 gene. The method according to any one of claims 1 to 4.

27. the flanking sequences are at least 17, at least 18, at least 19, at least 20, At least 21, at least 22, at least 23, at least 24, at least 25, a sequence of at least 30, at least 35, or at least 40 nucleotides in length; The method according to any one of claims 23 to 26.

28. The introduced nucleic acid sequence, or the first and second nucleic acid sequences, may contain a promoter. The method according to any one of claims 22 to 27, wherein the method does not include the step of:

29. The introduced nucleic acid sequence, or the first and second nucleic acid sequences, function as a promoter. Optionally, the promoter is operably linked to an EF-1α promoter (e.g., The method according to any one of claims 22 to 27, wherein the nucleic acid sequence is a sequence of a nucleic acid sequence selected from the group consisting of a nucleic acid sequence ...

30. The introduced nucleic acid sequence, or the first and second nucleic acid sequences, may be a vector, a trans introduced via transfection, lipid nanoparticles, or microinjection, 30. The method according to any one of claims 22 to 29.

31. The vector is a viral vector, and optionally, the viral vector The method of claim 30, wherein is an adeno-associated virus vector.

32. 1. A method for intralocus insertion of a TCR (such as a WT1-specific TCR), comprising: (i) inserting a TCR encoding ... comprising a guide sequence selected from 0, 95, 97, 98, 185, 214, and 218 (ii) a first guide RNA for inserting a TCR; (iii) an RNA-guided DNA binder; or a nucleic acid encoding an RNA-guided DNA binder; and (iii) a TCR (WT1-specific and delivering a donor nucleic acid molecule encoding a target TCR or other target gene to the target cell.

33. and further comprising delivering a second guide RNA comprising a sequence selected from SEQ ID NOs: 1-89.

33. The method of claim 32, comprising:

34. Claim 3, wherein the second guide RNA comprises a sequence selected from SEQ ID NOs: 179-184 3. The method according to claim 3.

35. The TCR is (a) SEQ ID NOs: 500, 503, 506, 509, 512, 515, 518, or 521, or at least 99%, at least an amino acid sequence with 95%, at least 90% identity; or (b) a TCRα chain polypeptide and a TCRα chain polypeptide selected from the following (i) to (viii): Rβ chain polypeptide, or at least 99%, at least 95%, or at least Amino acid sequences with at least 90% identity: i) SEQ ID NO: 501 and SEQ ID NO: 502; ii) SEQ ID NO: 504 and SEQ ID NO: 505; iii) SEQ ID NO: 507 and SEQ ID NO: 508; iv) SEQ ID NO: 510 and SEQ ID NO: 511; v) SEQ ID NO: 513 and SEQ ID NO: 514; vi) SEQ ID NO: 516 and SEQ ID NO: 517; vii) SEQ ID NO: 519 and SEQ ID NO: 520; viii) SEQ ID NO: 522 and SEQ ID NO: 523; The method according to any one of claims 32 to 34, wherein the TCR is a WT1-specific TCR comprising:

36. a. A guide RNA comprising: i. a guide sequence selected from SEQ ID NOs: 1-89; or ii. at least 17, at least 18 of the sequences selected from SEQ ID NOs: 1 to 89 , at least 19, or at least 20 consecutive nucleotides; or iii. At least 99%, at least to a sequence selected from SEQ ID NOs: 1 to 89 At least 98%, at least 97%, at least 96%, at least 95%, at least 94% , at least 93%, at least 92%, at least 91%, or at least 90% a guide sequence with identity; or iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; or v. a guide sequence comprising any one of SEQ ID NOs: 1-6; and, optionally, b. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A composition comprising:

37. Modifying the DNA sequence within the TRBC1 and / or TRBC2 genes in cells 37. The composition of claim 36, for use in

38. When inducing expression of the TRBC1 and / or TRBC2 genes in cells 38. The composition of claim 36 or claim 37, for use.

39. The method of claim 1, wherein the guide RNA comprises a sequence selected from any of SEQ ID NOs: 196 to 200.

39. The composition according to any one of claims 36 to 38.

40. a. A guide RNA comprising: i. a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218 a guide sequence of choice; or ii. SEQ ID NOs: 90-178, 185, and 213-218 At least 17, at least 18, at least 19, or fewer of the selected sequences at least 20 consecutive nucleotides; or iii. SEQ ID NOs: 90-178, 185, and 213-218 At least 99%, at least 98%, at least 97%, at least at least 96%, at least 95%, at least 94%, at least 93%, at least a guide sequence with 92%, at least 91%, or at least 90% identity; and teeth iv. any one of SEQ ID NOs: 90-113 and 213-218 a guide sequence comprising: v. a guide sequence comprising any one of SEQ ID NOs: 90-95; and, optionally, the law of nature b. RNA-guided DNA-binding agents or nucleic acids encoding RNA-guided DNA-binding agents 、 A composition comprising:

41. 41. The method of claim 40 for use in modifying a DNA sequence within a TRAC gene in a cell. The composition described.

42. 40 or 41, for use in reducing the expression of the TRAC gene in a cell.

42. The composition of claim 41.

43. The guide RNA is any one of SEQ ID NOs: 185 to 192 and 201 to 212 43. The composition of any one of claims 40 to 42, comprising a sequence selected from:

44. A cell modified by the method of any one of claims 1 to 43.

45. 45. The cell of claim 44, which has been modified in vitro.

46. 46. ​​The cell of claim 44 or claim 45, which is a T cell.

47. CD3 ＋ , CD4 ＋ and / or CD8 ＋ Any one of claims 44 to 46, which is a T cell. The cell described in any one of claims 1 to 4.

48. 48. The method according to any one of claims 44 to 47, wherein the cell is a mammalian cell, a primate cell, or a human cell. The cells described.

49. To generate T cells that lack endogenous T cell receptors and express non-endogenous T cell receptors. The cell according to any one of claims 44 to 48.

50. Claims for generating CAR-expressing T cells that lack endogenous T cell receptors. Item 49. The cell according to any one of Items 44 to 48.

51. After modification, CD3 － The cell according to any one of claims 44 to 50, which is a cell.

52. Before modification, it was CD3 ＋ cells, and after modification, CD3 － Any of claims 44 to 51 which becomes a cell The cell described in any one of claims 1 to 4.

53. and optionally further comprising one or more nucleic acid sequences encoding a polypeptide of interest. a. the one or more polypeptides of interest comprise a receptor; b. the one or more polypeptides of interest comprise an immune receptor; c. the one or more polypeptides of interest comprise a T cell receptor, and optionally: the T cell receptor is specific for WT1; or d. the one or more polypeptides of interest comprise a chimeric antigen receptor, and optionally wherein the chimeric antigen receptor is specific for WT1. A cell according to any one of claims 44 to 52.

54. The antibody further comprises one or more nucleic acid sequences encoding the α and β chains of the foreign T cell receptor. The cell according to any one of claims 44 to 53.

55. One or more nucleic acid sequences encoding the α and β chains of the foreign T cell receptor are 55. The cell of claim 54, wherein the nucleic acid sequence is within the TRAC locus of the mouse.

56. The antibody further comprises one or more nucleic acid sequences encoding the gamma and delta chains of the foreign T cell receptor. The cell according to any one of claims 44 to 55.

57. One or more nucleic acid sequences encoding the gamma and delta chains of the foreign T cell receptor are 57. The cell of claim 56, wherein the nucleic acid sequence is within the TRAC locus of the mouse.

58. The sequence of the TCR alpha chain is (i) SEQ ID NO: 501 or SEQ ID NO: 504; (ii) the sequence At least 99%, at least 95%, at least 10% of SEQ ID NO: 501 or SEQ ID NO: 504 At least 90%, at least 85%, at least 80%, at least 70%, or (of) a sequence having at least 60% identity; and SEQ ID NO: 501 or SEQ ID NO: 504 At least 20, at least 30, at least 40, at least 50, At least 60, at least 70, at least 80, at least 90, at least 10 0, at least 150, at least 200, or at least 250 amino acids and The sequence of the TCR β chain is (i) SEQ ID NO: 502 or SEQ ID NO: 505; (ii) the sequence At least 99%, at least 95%, at least 10% of SEQ ID NO: 502 or SEQ ID NO: 505 At least 90%, at least 85%, at least 80%, at least 70%, or (of) a sequence having at least 60% identity; and SEQ ID NO: 502 or SEQ ID NO: 505 At least 20, at least 30, at least 40, at least 50, At least 60, at least 70, at least 80, at least 90, at least 10 0, at least 150, at least 200, at least 250, or at least a contiguous subsequence of at least 300 amino acids, A cell according to any one of claims 54 to 57.

59. the TCR alpha chain is set forth in any of SEQ ID NOs: 500, 501, 503, and 504 and the β TCR chain is encoded by a nucleic acid sequence set forth in SEQ ID NOs: 500, 502, 503, and Any of claims 54 to 58, encoded by the nucleic acid sequence of any of claims 54 to 58 and 505. The cell described in claim 1.

60. The sequence of the TCRα chain is (i) SEQ ID NO: 513; (ii) a sequence at least equal to SEQ ID NO: 513 at least 99%, at least 95%, at least 90%, at least 85%, at least sequences with 80%, at least 70%, or at least 60% identity; and and at least 20, at least 30, at least 40, or at least 100 copies of SEQ ID NO:

513. At least 50, at least 60, at least 70, at least 80, at least 9 0, at least 100, at least 150, at least 200, or at least a contiguous subsequence of at least 250 amino acids, and the nucleic acid sequence of the TCR β chain is: (i) SEQ ID NO: 514; (ii) SEQ ID NO: 514 At least 99%, at least 95%, at least 90%, at least 85%, or at least a sequence having 80%, at least 70%, or (of) at least 60% identity with and at least 20, at least 30, at least 40 of SEQ ID NO: 514; At least 50, at least 60, at least 70, at least 80, at least At least 90, at least 100, at least 150, at least 200, at least a contiguous subsequence of at least 250, or at least 300 amino acids, A cell according to any one of claims 54 to 59.

61. wherein the α TCR chain is SEQ ID NO: 513 and the β TCR chain is SEQ ID NO:

514.

61. The cell of any one of claims 54 to 60.

62. 62. The method of claim 44, wherein the one or more genes are expressed from an endogenous promoter. A cell described in any one of claims 1 to 4.

63. The one or more genes are expressed from a heterologous promoter, and optionally, the heterologous promoter The promoter according to any one of claims 44 to 62, wherein the promoter is an EF-1α promoter. cell.

64. A cell population comprising the cells according to any one of claims 44 to 63, More than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, about 80% greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% are CD3 － Thin A cell cluster, a cell.

65. Approximately 90% of the population is CD3 － 65. The population of claim 64, wherein:

66. More than about 95% of the population is CD3 － 65. The population of claim 64, wherein:

67. More than 99% of the population is CD3 － 65. The population of claim 64, wherein:

68. A cell population comprising the cells of any one of claims 44 to 63, wherein about 50% of the population more than %, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, about More than 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% endogenous T cell receptors A group of cells that lacks a body.

69. 69. The population of claim 68, wherein greater than about 90% of the population lacks an endogenous T cell receptor.

70. 69. The population of claim 68, wherein greater than about 95% of the population lacks endogenous T cell receptors.

71. 69. The population of claim 68, wherein greater than about 99% of the population lacks endogenous T cell receptors.

72. The occurrence of the TRBC1, TRBC2 and / or TRAC genes in a population The expression is at least about 50%, at least about 55%, or less than that of the same unmodified cell population. At least about 60%, at least about 65%, at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95%, Any of claims 44 to 63, wherein the concentration is reduced by at least about 98%, or at least about 99%. A cell population comprising the cells described in any one of claims 1 to 4.

73. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 73. The population of claim 72, wherein the population is at least about 90%.

74. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 73. The population of claim 72, wherein the population is at least about 95%.

75. The expression of the TRBC1 gene, TRBC2 gene and / or TRAC gene is reduced 73. The population of claim 72, wherein the population is at least about 99%.

76. 76. A method according to any one of claims 64 to 75, wherein said decrease is a decrease in expression of the TRBC1 gene. Described population.

77. 77. A method according to any one of claims 64 to 76, wherein said decrease is a decrease in expression of the TRBC2 gene. Described population.

78. 78. The method according to any one of claims 64 to 77, wherein the decrease is a decrease in the expression of the TRAC gene. A group of people.

79. A cell population comprising cells according to any one of claims 44 to 78, wherein one of said populations 0-100%, for example 30-99% of the population, and / or a cell population having an indel in the TRAC gene.

80. 30-35%, 35-40%, 40-45%, 45-50%, 50-55% of the population 、５５～６０％、６０～６５％、６５～７０％、７０～７５％、７５～８０％、８０～８ 5%, 85-90%, 90-95%, or 95-99% of the TRBC1 gene, TRBC 80. The population of claim 79, having indels in the IL-2 gene and / or the TRAC gene.

81. 76 or claim 77, wherein the indel or insertion is in the TRBC1 gene.

80. The population described in claim 79.

82. 77 or claim 78, wherein the indel or insertion is in the TRBC2 gene.

80. The population described in claim 79.

83. 78 or claim 79, wherein the indel or insertion is in the TRAC gene.

80. The population according to paragraph 79.

84. wherein said composition results in editing of the TRBC1 and / or TRBC2 genes. The method according to any one of claims 1 to 4 or claims 9 to 43, or the method according to any one of claims 1 to 4 or claims 9 to 43 A composition for use according to any one of claims 9 to 43.

85. 44. The composition of any one of claims 4 to 43, wherein the composition effects editing of the TRAC gene. or a composition for use according to any one of claims 4 to 43.

86. Claims 9 to 43, wherein the composition results in editing of the TRBC and TRAC genes or a composition for use according to any one of claims 9 to 43. thing.

87. The edits are calculated as a percentage of the edited population (edit rate or indel rate), The method according to any one of claims 79 to 86 or any one of claims 79 to 86 Compositions for use.

88. the editing rate is 30 to 35%, 35 to 40%, 40 to 45%, or 45 to 50% of the population 、５０～５５％、５５～６０％、６０～６５％、６５～７０％、７０～７５％、７５～８ 0%, 80-85%, 85-90%, 90-95%, or 95-99%. The method according to any one of claims 84 to 87 or the method according to any one of claims 84 to 87 Composition of Use.

89. The composition comprises: any one of SEQ ID NOs: 179-184 and 196-200; or b. a guide sequence selected from any one of SEQ ID NOs: 1-89; or c. a guide sequence selected from SEQ ID NOs: 1-24; or d. a guide sequence selected from SEQ ID NOs: 1-6; 44. The method or composition of any one of claims 4 to 43, comprising an sgRNA comprising:

90. The composition comprises: any one of SEQ ID NOs: 186-192 and 201-212; or b. A sequence selected from any one of SEQ ID NOs: 90-178 and 213-218. a guide sequence; or c. a guide sequence selected from SEQ ID NOs: 90-113 and 213-218; or d. a guide sequence selected from SEQ ID NOs: 90-95; 44. The method or composition of any one of claims 4 to 43, comprising an sgRNA comprising:

91. The target sequence is selected from the group consisting of the TRBC1 gene, the TRBC2 gene and / or the TRAC gene. A sequence within the first exon, second exon, third exon, or fourth exon.

44. The method or composition according to any one of paragraphs 1 to 43.

92. The target sequence is the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene.

92. The method or composition of claim 91, wherein the sequence is within the first exon of

93. The target sequence is the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene.

92. The method or composition of claim 91, wherein the sequence is within the second exon of

94. The target sequence is a sequence of the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene.

92. The method or composition of claim 91, wherein the sequence is within the third exon.

95. The target sequence is the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene.

92. The method or composition of claim 91, wherein the sequence is within the fourth exon of

96. The target sequence is the human TRBC1 gene, the human TRBC2 gene and / or the human TRBC1 gene.

96. The method or composition of any one of claims 91 to 95, wherein the sequence is within the AC gene. 。

97. The guide sequence is a target sequence within the positive strand of TRBC1, TRBC2, and / or TRAC.

44. The method or composition of any one of claims 1 to 43, wherein the nucleic acid sequence is complementary to a target sequence. 。

98. The guide sequence may be a sequence encoding the TRBC1 gene, the TRBC2 gene, and / or the TRAC gene.

44. The method of claim 1, wherein the target sequence is complementary to a target sequence in the minus strand of the nucleic acid molecule. The method or composition described above.

99. The first guide sequence is The composition is complementary to a first target sequence in the positive strand of the TRBC1 gene. , against a second target sequence within the minus strand of the TRBC2 gene and / or the TRAC gene Any of claims 1 to 43 and claims 84 to 98, further comprising a second guide sequence complementary to said The method or composition according to any one of claims 1 to 4.

100. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178. and further comprising the nucleotide sequence of SEQ ID NO: 400, The nucleic acid sequence of claim 1 to claim 43 and claim 8 is a nucleic acid sequence comprising a nucleic acid sequence of claim 1, a nucleic acid sequence of claim 2, a nucleic acid sequence of claim 3, a nucleic acid sequence of claim 4, a nucleic acid sequence of claim 5, a nucleic acid sequence of claim 6, a nucleic acid sequence of claim 7, a nucleic acid sequence of claim 8, a nucleic acid sequence of claim 9, a nucleic acid sequence of claim 10, a nucleic acid sequence of claim 11, a nucleic acid 99. The method or composition of any one of claims 4 to 98.

101. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178. and further comprising the nucleotide sequence of SEQ ID NO: 401, The nucleic acid sequence of claim 1 to claim 43 or claim 8 is followed by a nucleic acid sequence at the 3' end of the guide sequence.

99. The method or composition of any one of claims 4 to 98.

102. The guide RNA is modified according to the pattern of SEQ ID NO: 300, and N is and any one of the guide sequences of SEQ ID NOs: 1 to 89, 84-98. The method or composition of any one of claims 84-98.

103. each N in SEQ ID NO:300 is any natural or non-natural nucleotide; The N forms a guide sequence, and the guide sequence directs Cas9 to the TRBC1 gene, TRBC 103. The method of claim 102, wherein the targeting gene is the IL-2 gene and / or the TRAC gene. composition.

104. the sgRNA is at least 99% specific to a sequence selected from SEQ ID NOs: 1-89, at least 98%, at least 97%, at least 98%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, or at least 9 104. The method of claim 100, comprising a guide sequence with 0% identity to the target gene. Method or composition.

105. Claims 1 to 43 and claim 8, wherein the guide RNA is contained in a viral vector. 4-104. The method or composition of any one of claims 4-104.

106. 105. The viral vector is an adeno-associated viral (AAV) vector.

2. The method or composition according to claim 1 .

107. The viral vector comprises a nucleic acid sequence of any of SEQ ID NOs: 613 to 632, or At least 99%, at least 95%, at least 90%, at least 85%, having at least 80%, at least 70%, or at least 60% identity 107. The method or composition of claim 105 or claim 106, comprising a nucleic acid sequence that

108. The guide RNA comprises at least one modification, 107. The method or composition of any one of claims 107 to 107.

109. The at least one modification is a 2'-O-methyl (2'-O-Me) modified nucleotide.

109. The method or composition of claim 108, comprising:

110. 108 or claim 109, comprising phosphorothioate (PS) linkages between nucleotides.

110. The method or composition of paragraph 109.

111. Any of claims 108 to 110, comprising 2'-fluoro (2'-F) modified nucleotides.

10. The method or composition according to claim 1 .

112. The guide RNA comprises a modification in one or more of the first five nucleotides at the 5' end.

112. The method or composition of any one of claims 108 to 111.

113. The guide RNA contains a modification in one or more of the last five nucleotides at the 3' end.

113. The method or composition of any one of claims 108 to 112.

114. A PS bond is contained between the first four nucleotides of the 5' end of the guide RNA.

114. A method or composition according to any one of paragraphs 108 to 113.

115. The guide RNA comprises a PS bond between the last four nucleotides at the 3' end of the guide RNA.

115. A method or composition according to any one of paragraphs 108 to 114.

116. The first three nucleotides at the 5' end of the guide RNA contain 2'-O-Me modified nucleotides.

116. The method or composition of any one of claims 108 to 115, comprising a oxide.

117. The last three nucleotides at the 3' end of the guide RNA are 2'-O-Me modified nucleotides.

117. The method or composition of any one of claims 108 to 116, comprising a oxide.

118. Claims 108-117, wherein the guide RNA comprises modified nucleotides of SEQ ID NO: 300 10. The method or composition of any one of claims 1 to 9.

119. The composition of any one of claims 108 to 118, wherein the composition further comprises a pharmaceutically acceptable excipient.

10. The method or composition of any one of claims 1 to 9.

120. Claims 1 to 43 and claim 44, wherein the composition further comprises an RNA-guided DNA binding agent. 84-119. A method or composition according to any one of claims 84-119.

121. The composition of the guide RNA and the RNA-guided DNA binding agent is a ribonucleoprotein (R NP) according to any one of claims 1 to 43 and claims 84 to 120 2. A method or composition of claim 1 .

122. 2. The method of claim 1 , wherein the composition further comprises an mRNA encoding an RNA-guided DNA-binding agent.

121. A method or composition according to any one of claims 1 to 43 and claims 84 to 120.

123. Claim 121 or Claim 122, wherein the RNA-guided DNA binding agent is Cas9.

2. The method or composition according to claim 1 .

124. 124. The method of claim 123, wherein the RNA-guided DNA binding agent is a Cas9 protein. Method or composition.

125. Claims 1 to 43, wherein the composition is a pharmaceutical preparation and further comprises a pharmaceutically acceptable carrier. and the method or composition of any one of claims 84 to 124.

126. A composition, formulation or population according to any one of claims 36 to 125 for the preparation of a medicament. , or the use of cells.

127. A method according to any one of claims 36 to 125 for the preparation of a medicament for use in the treatment of cancer. Use of the compositions, preparations, populations, or cells described herein.

128. Any one of claims 36 to 125 for the preparation of a medicament for use in immunotherapy of a subject. Use of a composition, preparation, population, or cell described in.

129. For the preparation of a medicament for use in the treatment of tumors overexpressing Wilms' tumor antigen (WT1), Use of a composition, preparation, population or cell according to any one of claims 36 to 125 for the treatment of For.

130. A composition according to any one of claims 36 to 125 for use in the treatment of a disease or disorder. an object, preparation, population, or cell.

131. A composition, formulation or collection of any one of claims 36 to 125 for use in immunotherapy. Group, or cell.

132. A composition or formulation according to any one of claims 36 to 125 for use in the treatment of cancer. A group, or cell.

133. 36 to 38, which are used in the treatment of tumors that overexpress Wilms' tumor antigen (WT1).

125. A composition, preparation, population, or cell according to any one of claims 125.

134. The administration of a composition, preparation, population or cell according to any one of claims 36 to 125 A method of treating a human or animal, including

135. The administration of a composition, preparation, population or cell according to any one of claims 36 to 125 20. A method for treating cancer in a human or animal, comprising:

136. The administration of a composition, preparation, population or cell according to any one of claims 36 to 125 A method of immunotherapy in humans or animals, comprising:

137. The administration of a composition, preparation, population or cell according to any one of claims 36 to 125 Treatment of tumors in humans or animals that overexpress Wilms' tumor antigen (WT1), including method.

138. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

1. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

139. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

2. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

140. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

3. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

141. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

4. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

142. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

5. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

143. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

6. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

144. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

7. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

145. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

8. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

146. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

9. 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 A method, use, composition, population or cell according to any one of the preceding claims.

147. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

10. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

148. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

11. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

149. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

12. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

150. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

13. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

151. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

14. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

152. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

15. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

153. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

16. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

154. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

17. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

155. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

18. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

156. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

19. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

157. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

20. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

158. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

21. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

159. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

22. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

160. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

23. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

161. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

24. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

162. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

25. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

163. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

26. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

164. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

27. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

165. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

28. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

166. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

29. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

167. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

30. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

168. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

31. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

169. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

32. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

170. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

33. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

171. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

34. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

172. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

35. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

173. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

36. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

174. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

37. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

175. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

38. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

176. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

39. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

177. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

40. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

178. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

41. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

179. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

42. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

180. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

43. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

181. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

44. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

182. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

45. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

183. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

46. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

184. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

47. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

185. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

48. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

186. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

49. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

187. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

50. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

188. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

51. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

189. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

52. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

190. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

53. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

191. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

54. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

192. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

55. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

193. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

56. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

194. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

57. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

195. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

58. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

196. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

59. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

197. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

60. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

198. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

61. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

199. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

62. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

200. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

63. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

201. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

64. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

202. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

65. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

203. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

66. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

204. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

67. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

205. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

68. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

206. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

69. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

207. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

70. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

208. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

71. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

209. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

72. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

210. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

73. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

211. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

74. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

212. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

75. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

213. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

76. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

214. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

77. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

215. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

78. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

216. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

79. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

217. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

80. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

218. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

81. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

219. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

82. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

220. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

83. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

221. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

84. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

222. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

85. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

223. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

86. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

224. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

87. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

225. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

88. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, population, or cell according to any one of claims 1 to 11.

226. The sequence of the guide RNA selected from SEQ ID NOs: 1 to 89 is SEQ ID NO:

89. Items 1 to 4, 9 to 39, 44 to 77, 79 to 82, 84, 86 to 89, or 91 to 137 2. The method, use, composition, or cell according to any one of claims 1 to 11.

227. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

185. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

228. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

90. 85-87, or 90-137. Or composition.

229. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

91. 85-87, or 90-137. Or composition.

230. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

92. 85-87, or 90-137. Or composition.

231. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

93. 85-87, or 90-137. Or composition.

232. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

94. 85-87, or 90-137. Or composition.

233. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

95. 85-87, or 90-137. Or composition.

234. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

96. 85-87, or 90-137. Or composition.

235. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

97. 85-87, or 90-137. Or composition.

236. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

98. 85-87, or 90-137. Or composition.

237. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, and 83 is SEQ ID NO:

99. 85-87, or 90-137. Or composition.

238. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

100. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

239. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

101. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

240. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 102 3, 85-87, or 90-137, the method, use, population, cell Or composition.

241. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

103. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

242. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

104. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

243. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

105. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

244. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

106. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

245. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

107. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

246. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

108. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

247. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

109. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

248. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

110. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

249. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

111. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

250. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

112. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

251. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

113. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

252. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

114. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

253. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

115. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

254. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

116. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

255. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

117. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

256. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

118. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

257. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

119. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

258. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

120. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

259. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

121. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

260. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 122 3, 85-87, or 90-137, the method, use, population, cell Or composition.

261. The sequence of the guide RNA selected from SEQ ID NOs: 90 to 178, and 213 to 218 is SEQ ID NO: 123, 5-87 or 90-137. composition.

262. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

124. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

263. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

125. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

264. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

126. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

265. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

127. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

266. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

128. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

267. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

129. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

268. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

130. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

269. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

131. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

270. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

132. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

271. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 133 3, 85-87, or 90-137, the method, use, population, cell Or composition.

272. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

134. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

273. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

135. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

274. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

136. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

275. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

137. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

276. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

138. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

277. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 139 3, 85-87, or 90-137, the method, use, population, cell Or composition.

278. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

140. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

279. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 141 3, 85-87, or 90-137, the method, use, population, cell Or composition.

280. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

142. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

281. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

143. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

282. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

144. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

283. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

145. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

284. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

146. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

285. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

147. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

286. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

148. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

287. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 149 3, 85-87, or 90-137, the method, use, population, cell Or composition.

288. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

150. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

289. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

151. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

290. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

152. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

291. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

153. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

292. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

154. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

293. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

155. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

294. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

156. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

295. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

157. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

296. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

158. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

297. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 159 3, 85-87, or 90-137, the method, use, population, cell Or composition.

298. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

160. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

299. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

161. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

300. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

162. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

301. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 163 3, 85-87, or 90-137, the method, use, population, cell Or composition.

302. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

164. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

303. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

165. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

304. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

166. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

305. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

167. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

306. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

168. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

307. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 169 3, 85-87, or 90-137, the method, use, population, cell Or composition.

308. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 170 3, 85-87, or 90-137, the method, use, population, cell Or composition.

309. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

171. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

310. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

172. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

311. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

173. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

312. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

174. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

313. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

175. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

314. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

176. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

315. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

177. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

316. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

178. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

317. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, claim 9 to claim 35, claim 40 to claim 75, claim 78 to claim 80, claim 8 is SEQ ID NO: 213 3, 85-87, or 90-137, the method, use, population, cell Or composition.

318. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

214. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

319. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

215. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

320. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

216. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

321. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claim 5 to claim 8, 9 to claim 35, 40 to claim 75, 78 to claim 80, 8 is SEQ ID NO:

217. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

322. The guide RNA is selected from SEQ ID NOs: 90 to 178, 185, and 213 to 218. The sequence of claims 5 to 8, 9 to 35, 40 to 75, 78 to 80, 8 is SEQ ID NO:

218. 3, 85-87, or 90-137, the method, use, population, cell Or composition.

Images