JPWO2020081613A5 - - Google Patents

Description

As used herein and in the appended claims, use of the singular forms "a,""an," and "the," and any singular form of any word, is express and clear. It should be noted that unless it is limited to a single referent, it includes multiple referents. As used herein, the term "include" and grammatical variations thereof are intended to be non-limiting, and a description of an item within a listing may refer to the listed item. does not exclude other similar items that may be substituted for or added to.

Various embodiments of the invention are presented below.
1. A method of modifying a DNA sequence within the TRBC1 gene and/or the TRBC2 gene, comprising:
delivering the composition to a 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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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;
A method, including
2. A method of modifying a DNA sequence, comprising:
delivering the composition to a cell;
The composition comprises
(a) a guide RNA comprising a sequence selected from:
i. a sequence comprising 15 contiguous nucleotides ± 10 nucleotides of the genome coordinates set forth in either Table 1 and/or Table 3 for SEQ ID NOs: 1-89 and SEQ ID NOs: 179-184;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence from (i);
iii. 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% of the sequences selected from (i) a guide sequence with % identity;
(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;
A method, including
3. A method of reducing expression of the TRBC1 gene and/or the TRBC2 gene, comprising:
delivering the composition to a 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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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. 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;
A method, including
4. A method of immunotherapy comprising:
administering the composition to the subject, its autologous cells, and/or allogeneic cells;
the composition comprising:
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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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. 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;
A method, including
5. A method of modifying a DNA sequence within the TRAC gene, comprising:
delivering the composition to a cell;
The composition comprises
a. A guide RNA comprising a sequence selected from:
i. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
iii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity;
iv. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; and
v. a guide sequence comprising any one of SEQ ID NOS: 90-95; or
b. 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;
A method, including
6. A method of modifying a DNA sequence, comprising:
delivering the composition to a cell;
The composition comprises
(a) a guide RNA comprising a sequence selected from:
i. A sequence comprising 15 contiguous nucleotides ± 10 nucleotides of the genome coordinates listed in any of Tables 2 and/or 3 for SEQ ID NOs:90-218;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence from (i);
iii. 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% of the sequences selected from (i) a guide sequence with % identity;
(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;
A method, including
7. A method of reducing expression of a TRAC gene comprising:
delivering the composition to a cell;
The composition comprises
a. A guide RNA comprising a sequence selected from:
i. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
iii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity;
iv. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; and
v. a guide sequence comprising any one of SEQ ID NOS: 90-95; or
b. 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;
A method, including
8. A method of immunotherapy comprising:
administering the composition to the subject, its autologous cells, and/or allogeneic cells;
the composition comprising:
a. A guide RNA comprising a sequence selected from:
i. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
iii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity;
iv. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; 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.); and optionally
c. an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;
A method, including
9. A method of modifying a DNA sequence within the TRBC1 gene, the TRBC2 gene and/or the TRAC gene, comprising:
delivering to the cell a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA binding agent or a nucleic acid encoding the 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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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;
containing an array selected from
The second guide RNA is
vi. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
vii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
viii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity;
ix. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; and
x. a guide sequence comprising any one of SEQ ID NOS: 90-95;
A method containing an array selected from .
10. A method of reducing expression of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene, comprising:
delivering to the cell a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA binding agent or a nucleic acid encoding the 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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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;
containing an array selected from
The second guide RNA is
i. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
iii. at least 99%; at least 98%; at least 97%; at least 96%; at least 95%; at least 94%; %, at least 92%, at least 91%, or at least 90% identity;
iv. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; and
v. a guide sequence comprising any one of SEQ ID NOS: 90-95;
A method containing an array selected from .
11. A method of immunotherapy comprising:
administering the composition to the subject, its autologous cells, or allogeneic cells;
said composition comprising a first guide RNA, a second guide RNA, and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;
The first guide RNA is
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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;
containing an array selected from
The second guide RNA is
vi. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
vii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218;
viii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity;
ix. a guide sequence comprising any one of SEQ ID NOs:90-113, SEQ ID NOs:185, and SEQ ID NOs:213-218; and
x. a guide sequence comprising any one of SEQ ID NOS: 90-95;
A method containing an array selected from .
12. 1. A method of expressing a heterologous immune receptor via an intralocus insertion at the TRAC locus, comprising:
delivering to the cell a first guide RNA, a second guide RNA, and an RNA-guided DNA binding agent or a nucleic acid encoding the 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, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89;
iii. 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 a guide sequence with at least 90% 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;
containing an array selected from
The second guide RNA is
vi. a guide sequence selected from SEQ ID NO: 90, 95, 97, 98, 185, 214, 218;
vii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 90, 95, 97, 98, 185, 214, and 218;
viii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOS: 90, 95, 97, 98, 185, 214, and 218 %, at least 92%, at least 91%, or at least 90% identity;
ix. a guide sequence comprising any one of SEQ ID NOs: 90, 95, 97, 185, and 214;
x. a guide sequence comprising any one of SEQ ID NOS: 90, 95, and 185; and
xi. a guide sequence comprising SEQ ID NO: 90 or 214;
A method containing an array selected from .
13. 13. The method of any one of 9-12 above, wherein 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.
14. 13. The method of any one of claims 9-12, wherein said first guide RNA comprises the sequence of SEQ ID NO:180 and said second guide RNA comprises the sequence of SEQ ID NO:186.
15. Any one of 9 to 12 above, wherein the first guide RNA comprises the sequence of any one of SEQ ID NO: 1, 2, 3, 5, 6, and the second guide RNA comprises the sequence of SEQ ID NO: 90. The method according to item 1.
16. Any one of 9 to 12 above, wherein the first guide RNA comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 5, 6, and the second guide RNA comprises the sequence of SEQ ID NO: 214. The method according to item 1.
17. 17. Any one of claims 9 to 16, wherein the first guide RNA, the second guide RNA, and the RNA-guided DNA binding agent or nucleic acid encoding the RNA-guided DNA binding agent are administered substantially simultaneously. the method of.
18. 18. A method according to any one of claims 9 to 17, wherein DNA sequences within the TRBC1 gene, the TRBC2 gene and/or the TRAC gene are modified simultaneously.
19. 19. The method of any one of claims 1-18, wherein an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent is introduced.
20. a. introducing a double-strand break (DSB) into the TRBC1 gene, the TRBC2 gene and/or the TRAC gene in the cell and/or subject; or
b. introducing a single-strand break (SSB) into the TRBC1 gene, the TRBC2 gene and/or the TRAC gene in the cell and/or subject; or
c. reducing the expression of TRBC1 gene, TRBC2 gene and/or TRAC gene in a cell and/or subject;
20. The method of any one of 1-19 above, further comprising
21. further comprising introducing a nucleic acid sequence encoding a polypeptide of interest, optionally
a. said one or more polypeptides of interest comprises a receptor;
b. said one or more polypeptides of interest comprise an immunoreceptor;
c. said one or more polypeptides of interest comprises a T cell receptor, and optionally said T cell receptor recognizes a cancer antigen;
d. said one or more polypeptides of interest comprises a WT1-specific T cell receptor, said T cell receptor recognizing WT1 or a fragment thereof;
e. said one or more polypeptides of interest comprise a chimeric antigen receptor, and optionally said chimeric antigen receptor recognizes a cancer antigen; or
f. said one or more polypeptides of interest comprise a WT1-specific chimeric antigen receptor, said chimeric antigen receptor recognizing WT1 or a fragment thereof;
21. The method according to any one of 1 to 20 above.
22. a. introducing TCRα and TCRβ chains;
b. introducing one or more nucleic acid sequences encoding TCRα and TCRβ chains;
c. introducing WT1-specific TCR α and β chains;
d. introducing one or more nucleic acid sequences encoding WT1-specific TCR α and β chains;
e. introducing a first TCR sequence selected from: (i) SEQ ID NO:501 or SEQ ID NO:504; (ii) at least 99%, at least 95%, at least 90% relative to SEQ ID NO:501 or SEQ ID NO:504; (iii) at least 20, at least 30, at least 40 of SEQ ID NO: 501 or SEQ ID NO: 504; a contiguous subsequence of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 250 amino acids;
and,
introducing a second TCR sequence selected from: (i) SEQ ID NO: 502 or SEQ ID NO: 505; (ii) at least 99%, at least 95%, at least 90% relative to SEQ ID NO: 502 or SEQ ID NO: 505; (iii) at least 20, at least 30, at least 40 of SEQ ID NO: 502 or SEQ ID NO: 505; contiguous subsequence of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, 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%, at least 95%, at least 90% relative to SEQ ID NO:510 or SEQ ID NO:513; (iii) at least 20, at least 30, at least 40 of SEQ ID NO: 510 or SEQ ID NO: 513; a contiguous subsequence of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 250 amino acids;
and,
introducing a second TCR sequence selected from: (i) SEQ ID NO:511 or SEQ ID NO:514; (ii) at least 99%, at least 95%, at least 90% relative to SEQ ID NO:511 or SEQ ID NO:514; (iii) at least 20, at least 30, at least 40 of SEQ ID NO: 511 or SEQ ID NO: 514; contiguous subsequence of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, 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);
h. introducing a nucleic acid sequence comprising a sequence encoding the second TCR sequence of (e) or (f);
i. introducing a nucleic acid sequence comprising the nucleic acid sequences of (g) and (h);
j. introducing a polypeptide selected from SEQ ID NO: 500, 503, 506, 509, 512, 515, 518, or 521, or an amino acid sequence having at least 99%, at least 95%, at least 90% identity thereto optionally by introducing a nucleic acid sequence encoding it;
k. introducing a TCR α chain polypeptide and a TCR β chain polypeptide selected from (i) to (vii) below, or an amino acid sequence having at least 99%, at least 95%, at least 90% identity thereto :
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. introducing nucleic acid sequences encoding the TCR α and TCR β chain polypeptides of (k);
21. The method of any one of the preceding paragraphs 1-20, further comprising
23. 23. The method of Claim 22, wherein said first nucleic acid sequence is flanked by sequences homologous to the first target locus.
24. 23. The method of claim 22, wherein said second nucleic acid sequence is flanked by sequences homologous to the second target locus.
25. 25. The method of any one of claims 23-24, wherein said first target locus is the TRAC gene, the TRBC1 gene, or the TRBC2 gene, eg the TRAC gene.
26. 26. The method of any one of claims 24-25, wherein the second target locus is the TRAC gene, the TRBC1 gene, or the TRBC2 gene, such as the TRBC1 gene or the TRBC2 gene.
27. 23-, wherein said 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, at least 30, at least 35, or at least 40 nucleotides in length. 27. The method of any one of clauses 26.
28. 28. The method of any one of claims 22-27, wherein said introduced nucleic acid sequence, or said first and second nucleic acid sequences, does not comprise a promoter.
29. 22, wherein said introduced nucleic acid sequence or said first and second nucleic acid sequences are operably linked to a promoter, and optionally said promoter is an EF-1α promoter (e.g., SEQ ID NO: 603). 28. The method of any one of clauses 1-27.
30. 30. Any one of claims 22-29, wherein said introduced nucleic acid sequence, or said first and second nucleic acid sequences are introduced via a vector, transfection, lipid nanoparticles, or microinjection. Method.
31. 31. The method of claim 30, wherein said vector is a viral vector, further optionally said viral vector is an adeno-associated viral vector.
32. A method for intralocus insertion of a TCR (such as a WT1-specific TCR) comprising: (i) inserting a TCR comprising a guide sequence selected from SEQ ID NOS: 90, 95, 97, 98, 185, 214, and 218 (ii) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; and (iii) a donor nucleic acid molecule encoding a TCR (such as a WT1-specific TCR). A method comprising delivering.
33. 33. The method of 32 above, further comprising delivering a second guide RNA comprising a sequence selected from SEQ ID NOs: 1-89.
34. 34. The method of Claim 33, wherein said second guide RNA comprises a sequence selected from SEQ ID NOS: 179-184.
35. The TCR is
(a) a polypeptide selected from SEQ ID NO: 500, 503, 506, 509, 512, 515, 518, or 521, or an amino acid sequence having at least 99%, at least 95%, at least 90% identity thereto ;or
(b) a TCR α chain polypeptide and a TCR β chain polypeptide selected from (i) to (viii) below, or an amino acid sequence having at least 99%, at least 95%, at least 90% identity thereto:
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,
35. The method of any one of 32-34 above, wherein the WT1-specific TCR comprises
36. a. Guide RNA containing:
i. a guide sequence selected from SEQ ID NOs: 1-89; or
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89; or
iii. 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 a guide sequence with at least 90% 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. an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;
A composition comprising:
37. 37. The composition according to 36 above, for use in modifying DNA sequences within the TRBC1 and/or TRBC2 genes in cells.
38. 38. The composition according to 36 or 37 above, for use in introducing expression of the TRBC1 gene and/or the TRBC2 gene in cells.
39. 39. The composition of any of 36-38 above, wherein said guide RNA comprises a sequence selected from any of SEQ ID NOS: 196-200.
40. a. Guide RNA containing:
i. a guide sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218; or
ii. at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:90-178, SEQ ID NOs:185, and SEQ ID NOs:213-218; or
iii. at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93 for sequences selected from SEQ ID NOs: 90-178, SEQ ID NOs: 185, and 213-218 %, at least 92%, at least 91%, or at least 90% identity; or
iv. a guide sequence comprising any one of SEQ ID NOs:90-113 and SEQ ID NOs:213-218; or
v. a guide sequence comprising any one of SEQ ID NOs: 90-95; and optionally
b. an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;
A composition comprising:
41. 41. The composition according to 40 above, for use in modifying the DNA sequence within the TRAC gene in a cell.
42. 42. The composition of 40 or 41 above for use in reducing expression of the TRAC gene in a cell.
43. 43. The composition of any of 40-42, wherein said guide RNA comprises a sequence selected from any of SEQ ID NOS: 185-192 and SEQ ID NOS: 201-212.
44. A cell modified by the method according to any one of 1 to 43 above.
45. 45. The cell of 44 above, modified in vitro.
46. 45. The cell according to 44 or 45 above, which is a T cell.
47. CD3 ⁺ , CD4 ⁺ , and/or CD8 ⁺ 47. The cell according to any one of 44-46 above, which is a T cell.
48. 48. The cell according to any of 44-47 above, which is a mammalian cell, a primate cell, or a human cell.
49. 49. A cell according to any one of 44 to 48 above for generating a T cell expressing a non-endogenous T cell receptor lacking an endogenous T cell receptor.
50. 49. A cell according to any of 44-48 above for generating CAR-expressing T cells that are deficient in endogenous T cell receptors.
51. CD3 after modification ^- 51. The cell according to any one of 44 to 50 above, which becomes a cell.
52. CD3 before modification ⁺ cells and after modification CD3 ^- 52. The cell according to any one of 44 to 51 above, which becomes a cell.
53. further comprising one or more nucleic acid sequences encoding the polypeptide of interest, optionally
a. said one or more polypeptides of interest comprises a receptor;
b. said one or more polypeptides of interest comprise an immunoreceptor;
c. said one or more polypeptides of interest comprises a T cell receptor, and optionally said T cell receptor is specific for WT1; or
d. wherein said one or more polypeptides of interest comprise a chimeric antigen receptor, and optionally said chimeric antigen receptor is specific for WT1;
The cell according to any one of 44 to 52 above.
54. 54. The cell of any of 44-53 above, further comprising one or more nucleic acid sequences encoding the α and β chains of an exogenous T cell receptor.
55. 55. The cell of claim 54, wherein the one or more nucleic acid sequences encoding the α and β chains of the exogenous T cell receptor are nucleic acid sequences within the TRAC locus of the genome.
56. 56. The cell of any of 44-55 above, further comprising one or more nucleic acid sequences encoding the gamma and delta chains of an exogenous T cell receptor.
57. 57. The cell of claim 56, wherein the one or more nucleic acid sequences encoding the gamma and delta chains of the exogenous T cell receptor are nucleic acid sequences within the genomic TRAC locus.
58. the sequence of said TCRα chain is (i) SEQ ID NO:501 or SEQ ID NO:504; (ii) at least 99%, at least 95%, at least 90%, at least 85%, at least 80% relative to SEQ ID NO:501 or SEQ ID NO:504 , at least 70%, or (of) at least 60% identity; and at least 20, at least 30, at least 40, at least 50, at least 60, at least a contiguous subsequence of 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 250 amino acids; and
the sequence of said TCR beta chain is (i) SEQ ID NO: 502 or SEQ ID NO: 505; , at least 70%, or (of) at least 60% identity; and at least 20, at least 30, at least 40, at least 50, at least 60, at least a contiguous subsequence of 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, or at least 300 amino acids;
58. The cell according to any one of 54-57 above.
59. said TCR α chain is encoded by a nucleic acid sequence according to any of SEQ ID NOS:500, 501, 503 and 504 and said βTCR chain is a nucleic acid sequence according to any of SEQ ID NOs:500, 502, 503 and 505 59. The cell according to any of 54-58 above, which is encoded by
60. (ii) at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, or (of and 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 a contiguous subsequence of 100, at least 150, at least 200, or at least 250 amino acids; and
(ii) at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70% relative to SEQ ID NO: 514, or ( of) sequences having at least 60% identity; and at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 of SEQ ID NO:514, a contiguous subsequence of at least 100, at least 150, at least 200, at least 250, or at least 300 amino acids;
59. The cell according to any one of 54-59 above.
61. 61. The cell according to any of the above 54-60, wherein said αTCR chain is SEQ ID NO:513 and said βTCR chain is SEQ ID NO:514.
62. 62. The cell of any of 44-61, wherein said one or more genes are expressed from an endogenous promoter.
63. 63. The cell of any of 44-62, wherein said one or more genes are expressed from a heterologous promoter, optionally said heterologous promoter is the EF-1α promoter.
64. A cell population comprising cells according to any one of 44 to 63 above, wherein more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, about 75 of the modified population %, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% is CD3 ^- A cell population, which is a cell.
65. >90% of said population is CD3 ^- 64 above, wherein the population is
66. >95% of said population is CD3 ^- 64 above, wherein the population is
67. >99% of said population is CD3 ^- 64 above, wherein the population is
68. A cell population comprising cells according to any of 44-63 above, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75% of the population , greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% lacking endogenous T-cell receptors.
69. 69. The population of 68 above, wherein greater than about 90% of the population lacks an endogenous T cell receptor.
70. 69. The population of 68 above, wherein greater than about 95% of the population lacks an endogenous T cell receptor.
71. 69. The population of 68 above, wherein greater than about 99% of the population lacks an endogenous T cell receptor.
72. expression of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene in the population is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% compared to the same unmodified cell population %, 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%, any of the above 44-63 A cell population containing the indicated cells.
73. 73. The population of 72 above, wherein the reduction in expression of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene is at least about 90%.
74. 73. The population of 72 above, wherein the reduction in expression of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene is at least about 95%.
75. 73. The population of 72 above, wherein the reduction in expression of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene is at least about 99%.
76. 76. The population according to any of 64-75 above, wherein said decrease is a decrease in TRBC1 gene expression.
77. 77. The population of any of 64-76 above, wherein said decrease is a decrease in TRBC2 gene expression.
78. 78. The population of any of 64-77 above, wherein said decrease is a decrease in TRAC gene expression.
79. A cell population comprising the cells according to any one of 44 to 78 above, wherein 10 to 100% of the population, for example 30 to 99% of the population, have an indel in the TRBC1 gene, the TRBC2 gene and/or the TRAC gene. have a cell population.
80. 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-35% of said population 79. The population of 79 above, wherein 80%, 80-85%, 85-90%, 90-95%, or 95-99% have indels in the TRBC1, TRBC2 and/or TRAC genes.
81. 80. The population of 76 or 79, wherein said indels or insertions are in the TRBC1 gene.
82. 80. The population of 77 or 79, wherein said indels or insertions are in the TRBC2 gene.
83. 80. The population of 78 or 79, wherein said indels or insertions are in the TRAC gene.
84. The method according to any one of 1 to 4 or 9 to 43, or the use according to any one of 1 to 4 or 9 to 43, wherein said composition effects editing of the TRBC1 gene and/or TRBC2 gene. composition.
85. 44. The method of any of 4-43, or the composition of use of any of 4-43, wherein said composition effects editing of the TRAC gene.
86. 44. The method of any of 9-43, or the composition of use of any of 9-43, wherein said composition effects editing of the TRBC and TRAC genes.
87. 87. A method according to any of 79-86 or a composition of use according to any of 79-86, wherein said editing is calculated as a percentage of the population that is edited (edit rate or indel rate).
88. The edit rate is 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70- The method of any of 84-87 or any of 84-87, wherein 75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-99%. A composition for use as described in .
89. the composition comprising:
a. any one of SEQ ID NOs: 179-184 and SEQ ID NOs: 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 of 4-43 above, comprising an sgRNA comprising
90. the composition comprising:
a. any one of SEQ ID NOs: 186-192 and SEQ ID NOs: 201-212; or
b. a guide sequence selected from any one of SEQ ID NOs:90-178 and SEQ ID NOs:213-218; or
c. a guide sequence selected from SEQ ID NOs:90-113 and SEQ ID NOs:213-218; or
d. a guide sequence selected from SEQ ID NOS: 90-95;
44. The method or composition of any of 4-43 above, comprising an sgRNA comprising
91. 44. The method or composition according to any one of 1 to 43 above, wherein said target sequence is a sequence within exon 1, exon 2, exon 3 or exon 4 of TRBC1 gene, TRBC2 gene and/or TRAC gene. thing.
92. 92. A method or composition according to claim 91, wherein said target sequence is a sequence within the first exon of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene.
93. 92. A method or composition according to claim 91, wherein said target sequence is a sequence within the second exon of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene.
94.
92. A method or composition according to claim 91, wherein said target sequence is a sequence within the third exon of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene.
95. 92. A method or composition according to claim 91, wherein said target sequence is a sequence within the 4th exon of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene.
96. 96. A method or composition according to any of the above 91-95, wherein said target sequence is a sequence within the human TRBC1 gene, the human TRBC2 gene and/or the human TRAC gene.
97. 44. The method or composition of any of claims 1-43, wherein said guide sequence is complementary to a target sequence within the plus strand of TRBC1, TRBC2 and/or TRAC.
98. 44. A method or composition according to any preceding 1 to 43, wherein said guide sequence is complementary to a target sequence within the minus strand of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene.
99. wherein the first guide sequence is complementary to a first target sequence within the positive strand of the TRBC1 gene, the TRBC2 gene and/or the TRAC gene, and the composition comprises 99. The method or composition of any of 1-43 and 84-98, further comprising a second guide sequence complementary to a second target sequence within.
100. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178, and further comprises a nucleotide sequence of SEQ ID NO: 400, wherein the nucleotide of SEQ ID NO: 400 is at the 3' end of the guide sequence. The method or composition of any of 1-43 and 84-98 that follow.
101. The guide RNA comprises a guide sequence selected from any one of SEQ ID NOs: 1 to 178, and further comprises a nucleotide sequence of SEQ ID NO: 401, wherein the nucleotide of SEQ ID NO: 401 is at the 3' end of the guide sequence. The method or composition of any of 1-43 or 84-98 that follow.
102. any one of 1-43 or 84-98 above, wherein said guide RNA is modified according to the pattern of SEQ ID NO: 300, and N, taken together, is any one of the guide sequences of SEQ ID NOS: 1-89 The described method or composition.
103. wherein each N within SEQ ID NO: 300 is any natural or non-natural nucleotide, said N forming a guide sequence, said guide sequence targeting Cas9 to the TRBC1 gene, the TRBC2 gene and/or the TRAC gene, above 102. The method or composition according to 102.
104. at least 99%, at least 98%, at least 97%, at least 98%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91% of the sequence wherein said sgRNA is selected from SEQ ID NOS: 1-89 %, or at least 90% identity.
105. 105. The method or composition of any of 1-43 and 84-104, wherein said guide RNA is contained in a viral vector.
106. 106. The method or composition of claim 105, wherein said viral vector is an adeno-associated virus (AAV) vector.
107. the viral vector is any of the nucleic acid sequences of SEQ ID NOS: 613-632, or at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, or (of) at least 107. A method or composition according to 105 or 106, comprising nucleic acid sequences having 60% identity.
108. 108. The method or composition of any of 1-43 and 84-107, wherein said guide RNA comprises at least one modification.
109. 109. The method or composition of claim 108, wherein said at least one modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides.
110. 109. The method or composition of 108 or 109, comprising phosphorothioate (PS) linkages between nucleotides.
111. The method or composition of any of 108-110 above, comprising a 2'-fluoro (2'-F) modified nucleotide.
112. 112. A method or composition according to any of the above 108-111, comprising modifications to one or more of the first 5 nucleotides of the 5' end of said guide RNA.
113. 113. A method or composition according to any of the above 108-112, comprising modifications to one or more of the last 5 nucleotides of the 3' end of said guide RNA.
114. 114. A method or composition according to any of the above 108-113, comprising a PS bond between the first four nucleotides of the 5' end of said guide RNA.
115. 115. The method or composition of any of 108-114 above, comprising a PS bond between the last four nucleotides of the 3' end of said guide RNA.
116. 116. The method or composition of any of 108-115 above, comprising 2'-O-Me modified nucleotides in the first three nucleotides of the 5' end of said guide RNA.
117. 117. The method or composition of any of 108-116 above, wherein the last three nucleotides of the 3' end of said guide RNA comprise 2'-O-Me modified nucleotides.
118. 118. The method or composition of any of 108-117 above, wherein said guide RNA comprises a modified nucleotide of SEQ ID NO:300.
119. 119. The method or composition of any of the above 108-118, wherein said composition further comprises a pharmaceutically acceptable excipient.
120. 120. The method or composition of any of 1-43 and 84-119, wherein said composition further comprises an RNA-guided DNA binding agent.
121. 121. The method or composition of any of clauses 1-43 and 84-120, wherein the guide RNA and RNA-guided DNA binding agent composition is comprised of a ribonucleoprotein (RNP).
122. 121. The method or composition of any of 1-43 and 84-120, wherein said composition further comprises an mRNA encoding an RNA-guided DNA binding agent.
123. 123. The method or composition of 121 or 122, wherein said RNA-guided DNA binding agent is Cas9.
124. 124. The method or composition of 123 above, wherein said RNA-guided DNA binding agent is a Cas9 protein.
125. 125. The method or composition of any of 1-43 and 84-124, wherein said composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
126. Use of the composition, formulation, population or cells of any of 36-125 above for the preparation of a medicament.
127. 126. Use of the composition, formulation, population or cells of any of 36-125 above for the preparation of a medicament for use in the treatment of cancer.
128. 126. Use of the composition, formulation, population or cells of any of 36-125 above for the preparation of a medicament for use in immunotherapy of a subject.
129. 126. Use of the composition, formulation, population or cells of any of 36-125 above for the preparation of a medicament for use in treating tumors overexpressing Wilm's Tumor Antigen (WT1).
130. 126. A composition, formulation, population or cell according to any of 36-125 above for use in the treatment of a disease or disorder.
131. 126. A composition, formulation, population or cell according to any of 36-125 above for use in immunotherapy.
132. 126. A composition, formulation, population or cell according to any of 36-125 above for use in the treatment of cancer.
133. 126. A composition, formulation, population or cell according to any of 36 to 125 above for use in the treatment of tumors overexpressing Wilm's tumor antigen (WT1).
134. A method of treating a human or animal comprising administration of a composition, formulation, population or cells according to any of 36-125 above.
135. A method of treating cancer in humans or animals comprising administration of the composition, formulation, population or cells of any of 36-125 above.
136. A method of human or animal immunotherapy comprising administration of a composition, formulation, population or cells according to any of 36-125 above.
137. A method of treating a tumor overexpressing Wilms tumor antigen (WT1) in a human or animal comprising administration of a composition, formulation, population or cell according to any of 36-125 above.
138. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 1 A method, use, composition, population or cell according to any one.
139. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:2 A method, use, composition, population or cell according to any one.
140. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:3 A method, use, composition, population or cell according to any one.
141. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 4 A method, use, composition, population or cell according to any one.
142. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 5 A method, use, composition, population or cell according to any one.
143. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 6 A method, use, composition, population or cell according to any one.
144. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 7 A method, use, composition, population or cell according to any one.
145. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:8 A method, use, composition, population or cell according to any one.
146. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:9 A method, use, composition, population or cell according to any one.
147. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 10 A method, use, composition, population or cell according to any one.
148. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 11 A method, use, composition, population or cell according to any one.
149. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 12 A method, use, composition, population or cell according to any one.
150. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 13 A method, use, composition, population or cell according to any one.
151. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 14 A method, use, composition, population or cell according to any one.
152. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 15 A method, use, composition, population or cell according to any one.
153. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 16 A method, use, composition, population or cell according to any one.
154. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 17 A method, use, composition, population or cell according to any one.
155. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 18 A method, use, composition, population or cell according to any one.
156. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 19 A method, use, composition, population or cell according to any one.
157. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 20 A method, use, composition, population or cell according to any one.
158. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 21 A method, use, composition, population or cell according to any one.
159. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 22 A method, use, composition, population or cell according to any one.
160. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 23 A method, use, composition, population or cell according to any one.
161. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 24 A method, use, composition, population or cell according to any one.
162. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 25 A method, use, composition, population or cell according to any one.
163. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 26 A method, use, composition, population or cell according to any one.
164. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 27 A method, use, composition, population or cell according to any one.
165. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 28 A method, use, composition, population or cell according to any one.
166. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 29 A method, use, composition, population or cell according to any one.
167. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:30 A method, use, composition, population or cell according to any one.
168. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 31 A method, use, composition, population or cell according to any one.
169. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 32 A method, use, composition, population or cell according to any one.
170. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 33 A method, use, composition, population or cell according to any one.
171. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 34 A method, use, composition, population or cell according to any one.
172. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 35 A method, use, composition, population or cell according to any one.
173. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 36 A method, use, composition, population or cell according to any one.
174. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 37 A method, use, composition, population or cell according to any one.
175. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:38 A method, use, composition, population or cell according to any one.
176. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:39 A method, use, composition, population or cell according to any one.
177. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 40 A method, use, composition, population or cell according to any one.
178. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 41 A method, use, composition, population or cell according to any one.
179. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 42 A method, use, composition, population or cell according to any one.
180. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 43 A method, use, composition, population or cell according to any one.
181. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 44 A method, use, composition, population or cell according to any one.
182. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 45 A method, use, composition, population or cell according to any one.
183. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 46 A method, use, composition, population or cell according to any one.
184. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 47 A method, use, composition, population or cell according to any one.
185. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 48 A method, use, composition, population or cell according to any one.
186. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 49 A method, use, composition, population or cell according to any one.
187. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 50 A method, use, composition, population or cell according to any one.
188. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 51 A method, use, composition, population or cell according to any one.
189. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 52 A method, use, composition, population or cell according to any one.
190. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 53 A method, use, composition, population or cell according to any one.
191. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 54 A method, use, composition, population or cell according to any one.
192. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 55 A method, use, composition, population or cell according to any one.
193. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 56 A method, use, composition, population or cell according to any one.
194. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 57 A method, use, composition, population or cell according to any one.
195. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 58 A method, use, composition, population or cell according to any one.
196. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 59 A method, use, composition, population or cell according to any one.
197. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 60 A method, use, composition, population or cell according to any one.
198. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 61 A method, use, composition, population or cell according to any one.
199. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 62 A method, use, composition, population or cell according to any one.
200. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 63 A method, use, composition, population or cell according to any one.
201. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 64 A method, use, composition, population or cell according to any one.
202. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 65 A method, use, composition, population or cell according to any one.
203. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 66 A method, use, composition, population or cell according to any one.
204. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 67 A method, use, composition, population or cell according to any one.
205. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO: 68 A method, use, composition, population or cell according to any one.
206. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 69 A method, use, composition, population or cell according to any one.
207. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 70 A method, use, composition, population or cell according to any one.
208. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 71 A method, use, composition, population or cell according to any one.
209. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 72 A method, use, composition, population or cell according to any one.
210. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 73 A method, use, composition, population or cell according to any one.
211. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 74 A method, use, composition, population or cell according to any one.
212. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 75 A method, use, composition, population or cell according to any one.
213. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 76 A method, use, composition, population or cell according to any one.
214. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 77 A method, use, composition, population or cell according to any one.
215. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 78 A method, use, composition, population or cell according to any one.
216. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 79 A method, use, composition, population or cell according to any one.
217. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:80 A method, use, composition, population or cell according to any one.
218. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:81 A method, use, composition, population or cell according to any one.
219. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 82 A method, use, composition, population or cell according to any one.
220. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:83 A method, use, composition, population or cell according to any one.
221. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 84 A method, use, composition, population or cell according to any one.
222. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 85 A method, use, composition, population or cell according to any one.
223. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:86 A method, use, composition, population or cell according to any one.
224. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 1-89 is SEQ ID NO: 87 A method, use, composition, population or cell according to any one.
225. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:88 A method, use, composition, population or cell according to any one.
226. Any of 1-4, 9-39, 44-77, 79-82, 84, 86-89, or 91-137 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 1-89 is SEQ ID NO:89 A method, use, composition or cell according to any one of the preceding claims.
227. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 185 , or the method, use, population, cell or composition of any of 90-137.
228. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:90 , or the method, use, population, cell or composition of any of 90-137.
229. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:91 , or the method, use, population, cell or composition of any of 90-137.
230. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:92 , or the method, use, population, cell or composition of any of 90-137.
231. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:93 , or the method, use, population, cell or composition of any of 90-137.
232. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:94 , or the method, use, population, cell or composition of any of 90-137.
233. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:95 , or the method, use, population, cell or composition of any of 90-137.
234. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:96 , or the method, use, population, cell or composition of any of 90-137.
235. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:97 , or the method, use, population, cell or composition of any of 90-137.
236. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:98 , or the method, use, population, cell or composition of any of 90-137.
237. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs:90-178, 185, and 213-218 is SEQ ID NO:99 , or the method, use, population, cell or composition of any of 90-137.
238. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 100 , or the method, use, population, cell or composition of any of 90-137.
239. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 101 , or the method, use, population, cell or composition of any of 90-137.
240. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 102 , or the method, use, population, cell or composition of any of 90-137.
241. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 103 , or the method, use, population, cell or composition of any of 90-137.
242. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 104 , or the method, use, population, cell or composition of any of 90-137.
243. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 105 , or the method, use, population, cell or composition of any of 90-137.
244. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 106 , or the method, use, population, cell or composition of any of 90-137.
245. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 107 , or the method, use, population, cell or composition of any of 90-137.
246. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 108 , or the method, use, population, cell or composition of any of 90-137.
247. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 109 , or the method, use, population, cell or composition of any of 90-137.
248. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 110 , or the method, use, population, cell or composition of any of 90-137.
249. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 111 , or the method, use, population, cell or composition of any of 90-137.
250. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 112 , or the method, use, population, cell or composition of any of 90-137.
251. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 113 , or the method, use, population, cell or composition of any of 90-137.
252. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 114 , or the method, use, population, cell or composition of any of 90-137.
253. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 115 , or the method, use, population, cell or composition of any of 90-137.
254. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 116 , or the method, use, population, cell or composition of any of 90-137.
255. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 117 , or the method, use, population, cell or composition of any of 90-137.
256. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 118 , or the method, use, population, cell or composition of any of 90-137.
257. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 119 , or the method, use, population, cell or composition of any of 90-137.
258. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 120 , or the method, use, population, cell or composition of any of 90-137.
259. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 121 , or the method, use, population, cell or composition of any of 90-137.
260. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 122 , or the method, use, population, cell or composition of any of 90-137.
261. 5-8, 9-35, 40-75, 78-80, 83, 85-87, or wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, and 213-218 is SEQ ID NO: 123 A method, use, population, cell or composition according to any of 90-137.
262. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 124 , or the method, use, population, cell or composition of any of 90-137.
263. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 125 , or the method, use, population, cell or composition of any of 90-137.
264. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 126 , or the method, use, population, cell or composition of any of 90-137.
265. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 127 , or the method, use, population, cell or composition of any of 90-137.
266. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 128 , or the method, use, population, cell or composition of any of 90-137.
267. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 129 , or the method, use, population, cell or composition of any of 90-137.
268. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 130 , or the method, use, population, cell or composition of any of 90-137.
269. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 131 , or the method, use, population, cell or composition of any of 90-137.
270. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 132 , or the method, use, population, cell or composition of any of 90-137.
271. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 133 , or the method, use, population, cell or composition of any of 90-137.
272. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 134 , or the method, use, population, cell or composition of any of 90-137.
273. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 135 , or the method, use, population, cell or composition of any of 90-137.
274. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 136 , or the method, use, population, cell or composition of any of 90-137.
275. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 137 , or the method, use, population, cell or composition of any of 90-137.
276. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 138 , or the method, use, population, cell or composition of any of 90-137.
277. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 139 , or the method, use, population, cell or composition of any of 90-137.
278. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 140 , or the method, use, population, cell or composition of any of 90-137.
279. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 141 , or the method, use, population, cell or composition of any of 90-137.
280. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 142 , or the method, use, population, cell or composition of any of 90-137.
281. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 143 , or the method, use, population, cell or composition of any of 90-137.
282. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 144 , or the method, use, population, cell or composition of any of 90-137.
283. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 145 , or the method, use, population, cell or composition of any of 90-137.
284. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 146 , or the method, use, population, cell or composition of any of 90-137.
285. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 147 , or the method, use, population, cell or composition of any of 90-137.
286. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 148 , or the method, use, population, cell or composition of any of 90-137.
287. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 149 , or the method, use, population, cell or composition of any of 90-137.
288. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 150 , or the method, use, population, cell or composition of any of 90-137.
289. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 151 , or the method, use, population, cell or composition of any of 90-137.
290. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 152 , or the method, use, population, cell or composition of any of 90-137.
291. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 153 , or the method, use, population, cell or composition of any of 90-137.
292. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 154 , or the method, use, population, cell or composition of any of 90-137.
293. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 155 , or the method, use, population, cell or composition of any of 90-137.
294. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 156 , or the method, use, population, cell or composition of any of 90-137.
295. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 157 , or the method, use, population, cell or composition of any of 90-137.
296. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 158 , or the method, use, population, cell or composition of any of 90-137.
297. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 159 , or the method, use, population, cell or composition of any of 90-137.
298. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 160 , or the method, use, population, cell or composition of any of 90-137.
299. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 161 , or the method, use, population, cell or composition of any of 90-137.
300. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 162 , or the method, use, population, cell or composition of any of 90-137.
301. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 163 , or the method, use, population, cell or composition of any of 90-137.
302. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 164 , or the method, use, population, cell or composition of any of 90-137.
303. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 165 , or the method, use, population, cell or composition of any of 90-137.
304. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 166 , or the method, use, population, cell or composition of any of 90-137.
305. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 167 , or the method, use, population, cell or composition of any of 90-137.
306. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 168 , or the method, use, population, cell or composition of any of 90-137.
307. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 169 , or the method, use, population, cell or composition of any of 90-137.
308. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 170 , or the method, use, population, cell or composition of any of 90-137.
309. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 171 , or the method, use, population, cell or composition of any of 90-137.
310. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 172 , or the method, use, population, cell or composition of any of 90-137.
311. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 173 , or the method, use, population, cell or composition of any of 90-137.
312. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 174 , or the method, use, population, cell or composition of any of 90-137.
313. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 175 , or the method, use, population, cell or composition of any of 90-137.
314. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 176 , or the method, use, population, cell or composition of any of 90-137.
315. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 177 , or the method, use, population, cell or composition of any of 90-137.
316. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOS: 90-178, 185, and 213-218 is SEQ ID NO: 178 , or the method, use, population, cell or composition of any of 90-137.
317. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 213 , or the method, use, population, cell or composition of any of 90-137.
318. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 214 , or the method, use, population, cell or composition of any of 90-137.
319. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 215 , or the method, use, population, cell or composition of any of 90-137.
320. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 216 , or the method, use, population, cell or composition of any of 90-137.
321. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 217 , or the method, use, population, cell or composition of any of 90-137.
322. 5-8, 9-35, 40-75, 78-80, 83, 85-87 above, wherein the sequence of said guide RNA selected from SEQ ID NOs: 90-178, 185, and 213-218 is SEQ ID NO: 218 , or the method, use, population, cell or composition of any of 90-137.

Claims (28)

(a) a TRAC guide RNA and optionally a TRBC1/2 guide RNA; (b) a nucleic acid encoding an RNA-guided DNA binder or an RNA-guided DNA binder; and (c) a nucleic acid encoding a heterologous immune receptor. , a composition for use in reducing expression of endogenous T-cell receptors and inserting heterologous immune receptors, comprising:
The TRAC guide RNA is:
i. a guide sequence that is SEQ ID NO: 90, or a guide sequence that is 18 or 19 contiguous nucleotides at the 3' end of SEQ ID NO: 90; or ii. a guide sequence that is SEQ ID NO:95, or a guide sequence that is 18 or 19 contiguous nucleotides at the 5' end of SEQ ID NO:95; or iii. a guide sequence that is SEQ ID NO: 185, or a guide sequence that is 18 or 19 contiguous nucleotides at the 5' end of SEQ ID NO: 185; or iv. a guide sequence that is SEQ ID NO:214 or a guide sequence that is 18 or 19 contiguous nucleotides at the 3' end of SEQ ID NO:214; or v. a guide sequence selected from SEQ ID NOs: 91-94, 96-98, 100, 102-178, 213, and 215-218, or SEQ ID NOs: 91-94, 96-98, 100, 102-178, 213, and 215 a guide sequence that is at least 18 or 19 contiguous nucleotides of a sequence selected from -218;
and, if present, said TRBC1/2 guide RNA is:
i. a guide sequence that is SEQ ID NO:2, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO:2; or ii. a guide sequence selected from SEQ ID NOS: 1-89, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89; or iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89; or iv. a guide sequence selected from SEQ ID NOs: 1-24; or v. A composition comprising a guide sequence selected from SEQ ID NOs: 1-6. A cell modified by the composition of claim 1. A cell population comprising one or more cells according to claim 2. 4. The cell population of claim 3, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80% of the cells of the population , greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% lacking endogenous T-cell receptors. 5. The cell population of claim 3 or 4, wherein more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, about 80% of the cells of the population %, greater than about 85%, or greater than about 90% express heterologous immune receptors. A cell population according to any one of claims 3-5 for use in treating cancer in a human or animal subject. 7. The cell population of claim 6, wherein said cancer is characterized by a tumor overexpressing Wilm's tumor antigen (WT1). 2. The composition of claim 1, wherein the nucleic acid encoding said heterologous immune receptor is flanked by sequences homologous to the TRAC locus. 9. The composition of claim 1 or 8, wherein said TRAC guide RNA comprises SEQ ID NO:90. 10. The composition of any one of claims 1, 8 and 9, comprising TRBC1/2 guide RNA, wherein said TRBC1/2 guide RNA comprises SEQ ID NO:2. 10. The composition of any one of claims 1, 8 and 9, comprising TRBC1/2 guide RNA, wherein said TRAC guide RNA comprises SEQ ID NO:90 and said TRBC1/2 guide RNA comprises SEQ ID NO:2. A composition comprising: 11. The composition of any one of claims 1, 8 and 10, wherein the TRAC guide RNA is:
i. a guide sequence that is SEQ ID NO:95 or a guide sequence that is 18 or 19 contiguous nucleotides at the 5' end of SEQ ID NO:95; or ii. a guide sequence that is SEQ ID NO:214, or a guide sequence that is 18 or 19 contiguous nucleotides at the 3' end of SEQ ID NO:214; or iii. a guide sequence selected from SEQ ID NOs: 91-94, 102-109, 111-113, 115-178, 213, and 215-218, or SEQ ID NOs: 91-94, 102-109, 111-113, 115-178, 213, and a guide sequence that is at least 18 or 19 contiguous nucleotides of a sequence selected from 215-218. The composition of any one of claims 1 and 8-12, wherein said heterologous immune receptor is a T-cell receptor. 14. The composition of claim 13, wherein said T cell receptor recognizes a cancer antigen. 15. The composition of claim 13 or 14, wherein said T-cell receptor is a WT1-specific T-cell receptor that recognizes WT1 or a fragment thereof. The composition of any one of claims 1 and 8-15, wherein said heterologous immune receptor is a chimeric antigen receptor. 17. The composition of claim 16, wherein said chimeric antigen receptor recognizes a cancer antigen. A composition according to any one of claims 1 and 8-17, comprising a TRBC1/2 guide RNA. The composition of any one of claims 1 and 8-18, wherein the RNA-guided DNA binding agent is Cas9. said TRAC guide RNA and, if present, said TRBC1/2 guide RNA, said nucleic acid encoding said RNA-guided DNA binder or RNA-guided DNA binder, and nucleic acid encoding said heterologous immune receptor in a vector; 20. The composition according to any one of claims 1 and 8-19, concomitantly used by a method comprising transfection, lipid nanoparticles or microinjection. (a) a TRAC guide RNA and a TRBC1/2 guide RNA; and (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. A composition for use comprising:
The TRAC guide RNA is:
i. guide sequences selected from SEQ ID NOs: 90-98, 100-178, 185 and 213-218, or at least 17, 18 of the sequences selected from SEQ ID NOs: 90-98, 100-178, 185 and 213-218 , 19, or 20 contiguous nucleotides; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOS: 90-98, 100-178, 185 and 213-218; or iii. a guide sequence selected from SEQ ID NOs: 90-95, 185 and 214, or at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-95, 185 and 214 a guide sequence; or iv. comprising a guide sequence of SEQ ID NO: 90, said TRBC1/2 guide RNA comprising:
i. a guide sequence that is SEQ ID NO:2, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO:2; or ii. a guide sequence selected from SEQ ID NOS: 1-89, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89; or iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89; or iv. a guide sequence selected from SEQ ID NOs: 1-24; or v. comprising a guide sequence selected from SEQ ID NOS: 1-6, or said TRAC guide RNA comprising:
i. guide sequences selected from SEQ ID NOs: 90-178, 185 and 213-218, or at least 17, 18, 19, or 20 of the sequences selected from SEQ ID NOs: 90-178, 185 and 213-218 a guide sequence that is contiguous nucleotides; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOS: 90-178, 185 and 213-218; or iii. a guide sequence selected from SEQ ID NOs: 90-95, 185 and 214, or at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-95, 185 and 214 a guide sequence; or iv. comprising a guide sequence of SEQ ID NO: 90, said TRBC1/2 guide RNA comprising:
i. a guide sequence selected from SEQ ID NOs: 1 and 3-89, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 and 3-89; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1 and 3-89; or iii. a guide sequence selected from SEQ ID NOs: 1 and 3-24; or iv. A composition comprising a guide sequence selected from SEQ ID NOs: 1 and 3-6. A cell modified by the composition of claim 21. 23. A cell population comprising one or more cells of claim 22. 24. The cell population of claim 23, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80% of the cells of the population , greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% lacking endogenous T-cell receptors. The composition of any one of claims 21-24, further comprising a nucleic acid encoding a heterologous immune receptor. 26. The composition of any one of claims 21-25, wherein the TRAC guide RNA comprises SEQ ID NO:90. 27. The composition of any one of claims 21-26, wherein said TRBC1/2 guide RNA comprises SEQ ID NO:2. A composition comprising TRAC guide RNA and TRBC1/2 guide RNA,
The TRAC guide RNA is:
i. guide sequences selected from SEQ ID NOs: 90-98, 100-178, 185 and 213-218, or at least 17, 18 of the sequences selected from SEQ ID NOs: 90-98, 100-178, 185 and 213-218 , 19, or 20 contiguous nucleotides; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOS: 90-98, 100-178, 185 and 213-218; or iii. a guide sequence selected from SEQ ID NOs: 90-95, 185 and 214, or at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-95, 185 and 214 a guide sequence; or iv. comprising a guide sequence of SEQ ID NO: 90, said TRBC1/2 guide RNA comprising:
i. a guide sequence that is SEQ ID NO:2, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO:2; or ii. a guide sequence selected from SEQ ID NOS: 1-89, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 1-89; or iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89; or iv. a guide sequence selected from SEQ ID NOs: 1-24; or v. comprising a guide sequence selected from SEQ ID NOS: 1-6, or said TRAC guide RNA comprising:
i. guide sequences selected from SEQ ID NOs: 90-178, 185 and 213-218, or at least 17, 18, 19, or 20 of the sequences selected from SEQ ID NOs: 90-178, 185 and 213-218 a guide sequence that is contiguous nucleotides; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOS: 90-178, 185 and 213-218; or iii. a guide sequence selected from SEQ ID NOs: 90-95, 185 and 214, or at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-95, 185 and 214 a guide sequence; or iv. comprising a guide sequence of SEQ ID NO: 90, said TRBC1/2 guide RNA comprising:
i. a guide sequence selected from SEQ ID NOs: 1 and 3-89, or a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 and 3-89; or ii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1 and 3-89; or iii. a guide sequence selected from SEQ ID NOs: 1 and 3-24; or iv. A composition comprising a guide sequence selected from SEQ ID NOs: 1 and 3-6.