Methods, compositions and components for CRISPR-Cas9 editing of TGFBR2 in T cells for immunotherapy
CRISPR/CAS-related genome editing systems, compositions and methods for targeting the TGFBR2 locus, as well as cells edited using these systems, compositions and methods are provided.
1 . A genome editing system comprising:
a guide RNA (gRNA) comprising a targeting domain that is complementary with a target sequence of a Transforming Growth Factor β Receptor II (TGFBR2) gene; and
an RNA-guided nuclease,
wherein the targeting domain comprises a nucleotide sequence that is identical to, or differs by no more than 3 nucleotides from, a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO: 5041;
(b) SEQ ID NO: 5042;
(c) SEQ ID NO: 5047;
(d) SEQ ID NO: 5050;
(e) SEQ ID NO: 5052;
(f) SEQ ID NO: 5092; and
(g) SEQ ID NO: 5093.
2 . The genome editing system of claim 1 , wherein the target sequence of the TGFBR2 gene comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.
3 . The genome editing system of claim 1 , wherein the RNA-guided nuclease is a Cas9 nuclease.
4 . The genome editing system of claim 3 , wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease.
5 . The genome editing system of claim 3 , wherein the Cas9 nuclease recognizes a Protospacer Adjacent Motif (PAM) of NGG.
6 . The genome editing system of claim 1 , wherein the RNA-guided nuclease is a Staphylococcus aureus Cas9 nuclease.
7 . The genome editing system of claim 6 , wherein the RNA-guided nuclease is a mutant Cas9 nuclease.
8 . The genome editing system of claim 1 , wherein the gRNA is a modular gRNA or a chimeric gRNA.
9 . The genome editing system of claim 1 , wherein the targeting domain comprises at least 18 contiguous nucleotides that are complementary to the TGFBR2 gene.
10 . The genome editing system of claim 1 , wherein the genome editing system comprises two, three or four gRNAs.
11 . A composition comprising a guide RNA (gRNA) or a vector comprising a polynucleotide encoding the gRNA, wherein the gRNA comprises a targeting domain that is complementary with a target sequence of a Transforming Growth Factor β Receptor II (TGFBR2) gene, and wherein the targeting domain comprises a nucleotide sequence that is identical to, or differs by no more than 3 nucleotides from, a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO: 5041;
(b) SEQ ID NO: 5042;
(c) SEQ ID NO: 5047;
(d) SEQ ID NO: 5050;
(e) SEQ ID NO: 5052;
(f) SEQ ID NO: 5092; and
(g) SEQ ID NO: 5093.
12 . The composition of claim 11 , wherein:
the target sequence of the TGFBR2 gene comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.
13 . The composition of claim 11 , further comprising a Cas9 nuclease or a polynucleotide encoding the Cas9 nuclease.
14 . The composition of claim 13 , wherein:
the Cas9 nuclease is a Staphylococcus aureus Cas9 nuclease, optionally wherein the Staphylococcus aureus Cas9 nuclease recognizes a Protospacer Adjacent Motif (PAM) of either NNNRRT or NNNRRV; or
the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease, optionally wherein the Streptococcus pyogenes Cas9 nuclease recognizes a Protospacer Adjacent Motif (PAM) of NGG.
15 . The composition of claim 13 , wherein the composition further comprises one or both of a wild-type Cas9 nuclease and a mutant Cas9 nuclease.
16 . The composition of claim 11 , wherein the targeting domain comprises at least 18 contiguous nucleotides that are complementary to the TGFBR2 gene.
17 . The composition of claim 11 , wherein the composition comprises one, two, three, or four gRNAs.
18 . The composition of claim 11 , wherein the vector is a viral vector.
19 . The composition of claim 18 , wherein the viral vector is an adeno-associated virus (AAV) vector or a lentivirus (LV) vector.