Genome editing of a Rosa locus using zinc-finger nucleases
First Claim
1. An isolated fusion protein comprising a nuclease domain and an engineered zinc finger DNA-binding domain, wherein the engineered zinc finger DNA-binding domain binds to a target site in a rat Rosa gene and the engineered zinc finger DNA-binding domain comprises four, five or six zinc finger DNA recognition regions designated and ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus and further wherein the engineered zinc finger DNA-binding domain comprises the zinc finger DNA recognition regions selected from the group consisting of:
- (i) F1;
DRSDLSR (SEQ ID NO;
38);
F2;
RSDDLTR (SEQ ID NO;
39);
F3;
TSGHLSR (SEQ ID NO;
40);
F4;
RSDNLSV (SEQ ID NO;
41); and
F5;
RSANLTR (SEQ ID NO;
42);
(ii) F1;
QSDHLTK (SEQ ID NO;
43);
F2;
NSSNLSR (SEQ ID NO;
44);
F3;
RSDHLTK (SEQ ID NO;
45);
F4;
NSDHLSR (SEQ ID NO;
46); and
F5;
RSDHLSR (SEQ ID NO;
47);
(iii) F1;
RSDHLSE (SEQ ID NO;
48);
F2;
RSAALAR (SEQ ID NO;
49);
F3;
RSDHLST (SEQ ID NO;
50);
F4;
QNAHRIT (SEQ ID NO;
51); and
F5;
RSAVLSE (SEQ ID NO;
52);
(iv) F1;
QSGDLTR (SEQ ID NO;
17);
F2;
TSGSLTR (SEQ ID NO;
18);
F3;
RSANLTR (SEQ ID NO;
42);
F4;
RSDHLTK (SEQ ID NO;
45); and
F5;
NSDHLSR (SEQ ID NO;
46);
(v) F1;
RSANLTR(SEQ ID NO;
42);
F2;
QSGDLTR (SEQ ID NO;
17);
F3;
QSGDLTR (SEQ ID NO;
17);
F4;
RSANLAR (SEQ ID NO;
53); and
F5;
RSDNLRE (SEQ ID NO;
54);
(vi) F1;
RSDHLST (SEQ ID NO;
50);
F2;
DNRDRIK (SEQ ID NO;
55);
F3;
RSDTLSE (SEQ ID NO;
56);
F4;
QSSHLAR (SEQ ID NO;
57); and
F5;
QNAHRKT (SEQ ID NO;
22);
(vii) F1;
QSGDLTR (SEQ ID NO;
17);
F2;
QSGDLTR (SEQ ID NO;
17);
F3;
RSDNLTR (SEQ ID NO;
58);
F4;
RSDNLSE (SEQ ID NO;
21); and
F5;
QNAHRKT (SEQ ID NO;
22);
(viii) F1;
DRSDLSR (SEQ ID NO;
38);
F2;
RSDHLST (SEQ ID NO;
50);
F3;
DNRDRIK (SEQ ID NO;
55);
F4;
RSDTLSE (SEQ ID NO;
56); and
F5;
QSSHLAR (SEQ ID NO;
57);
(ix) F1;
QSGDLTR (SEQ ID NO;
17);
F2;
RSDNLTR (SEQ ID NO;
58);
F3;
RSDNLSE (SEQ ID NO;
21);
F4;
QNAHRKT (SEQ ID NO;
22);
F5;
RSDHLSE (SEQ ID NO;
48); and
F6;
TSSTRKT (SEQ ID NO;
59);
(x) F1;
TSGNLTR(SEQ ID NO;
60);
F2;
QSGNLAR (SEQ ID No;
61);
F3;
RSDALSV (SEQ ID NO;
62);
F4;
DSSHRTR (SEQ ID NO;
63); and
F5;
RSDVLSE (SEQ ID NO;
64)(xi) F1;
RSDNLSE (SEQ ID NO;
21);
F2;
QNAHRKT (SEQ ID NO;
22);
F3;
RSDHLSE (SEQ ID NO;
48);
F4;
TSSTRKT (SEQ ID NO;
59); and
F5;
TSGHLSR (SEQ ID NO;
40);
(xii) F1;
TSGNLTR (SEQ ID NO;
60);
F2;
QSGNLAR (SEQ ID NO;
61);
F3;
RSDALSV (SEQ ID NO;
62); and
F4;
DSSHRTR (SEQ ID NO;
63);
(xiii) F1;
QRSNLVR (SEQ ID NO;
65);
F2;
RSDHLTQ (SEQ ID NO;
66);
F3;
QSGHLQR (SEQ ID NO;
67); and
F4;
DRSHLAR (SEQ ID NO;
68);
(xiv) F1;
RSDVLSE (SEQ ID NO;
64);
F2;
QRNHRTT (SEQ ID NO;
69);
F3;
TKRSLIE (SEQ ID NO;
70);
F4;
TSSNLSR (SEQ ID NO;
71);
F5;
RSDDLSK (SEQ ID NO;
25); and
F6;
DNRDRIK (SEQ ID NO;
55);
(xv) F1;
RSDHLSA (SEQ ID NO;
72);
F2;
QSGHLSR (SEQ ID NO;
24);
F3;
RSDHLSR (SEQ ID NO;
47);
F4;
QNDNRIK (SEQ ID NO;
73); and
F5;
QSGNLAR (SEQ ID NO;
61);
(xvi) F1;
NNRDLIN (SEQ ID NO;
74);
F2;
TSSNLSR (SEQ ID NO;
71);
F3;
RSDVLSE (SEQ ID NO;
64);
F4;
QRNHRTT (SEQ ID NO;
69);
F5;
TKRSLIE (SEQ ID NO;
70); and
F6;
TSSNLSR (SEQ ID NO;
71).
2 Assignments
0 Petitions
Accused Products
Abstract
Disclosed herein are methods and compositions for genome editing of a Rosa locus, using fusion proteins comprising a zinc-finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.
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Citations
20 Claims
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1. An isolated fusion protein comprising a nuclease domain and an engineered zinc finger DNA-binding domain, wherein the engineered zinc finger DNA-binding domain binds to a target site in a rat Rosa gene and the engineered zinc finger DNA-binding domain comprises four, five or six zinc finger DNA recognition regions designated and ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus and further wherein the engineered zinc finger DNA-binding domain comprises the zinc finger DNA recognition regions selected from the group consisting of:
-
(i) F1;
DRSDLSR (SEQ ID NO;
38);F2;
RSDDLTR (SEQ ID NO;
39);F3;
TSGHLSR (SEQ ID NO;
40);F4;
RSDNLSV (SEQ ID NO;
41); andF5;
RSANLTR (SEQ ID NO;
42);(ii) F1;
QSDHLTK (SEQ ID NO;
43);F2;
NSSNLSR (SEQ ID NO;
44);F3;
RSDHLTK (SEQ ID NO;
45);F4;
NSDHLSR (SEQ ID NO;
46); andF5;
RSDHLSR (SEQ ID NO;
47);(iii) F1;
RSDHLSE (SEQ ID NO;
48);F2;
RSAALAR (SEQ ID NO;
49);F3;
RSDHLST (SEQ ID NO;
50);F4;
QNAHRIT (SEQ ID NO;
51); andF5;
RSAVLSE (SEQ ID NO;
52);(iv) F1;
QSGDLTR (SEQ ID NO;
17);F2;
TSGSLTR (SEQ ID NO;
18);F3;
RSANLTR (SEQ ID NO;
42);F4;
RSDHLTK (SEQ ID NO;
45); andF5;
NSDHLSR (SEQ ID NO;
46);(v) F1;
RSANLTR(SEQ ID NO;
42);F2;
QSGDLTR (SEQ ID NO;
17);F3;
QSGDLTR (SEQ ID NO;
17);F4;
RSANLAR (SEQ ID NO;
53); andF5;
RSDNLRE (SEQ ID NO;
54);(vi) F1;
RSDHLST (SEQ ID NO;
50);F2;
DNRDRIK (SEQ ID NO;
55);F3;
RSDTLSE (SEQ ID NO;
56);F4;
QSSHLAR (SEQ ID NO;
57); andF5;
QNAHRKT (SEQ ID NO;
22);(vii) F1;
QSGDLTR (SEQ ID NO;
17);F2;
QSGDLTR (SEQ ID NO;
17);F3;
RSDNLTR (SEQ ID NO;
58);F4;
RSDNLSE (SEQ ID NO;
21); andF5;
QNAHRKT (SEQ ID NO;
22);(viii) F1;
DRSDLSR (SEQ ID NO;
38);F2;
RSDHLST (SEQ ID NO;
50);F3;
DNRDRIK (SEQ ID NO;
55);F4;
RSDTLSE (SEQ ID NO;
56); andF5;
QSSHLAR (SEQ ID NO;
57);(ix) F1;
QSGDLTR (SEQ ID NO;
17);F2;
RSDNLTR (SEQ ID NO;
58);F3;
RSDNLSE (SEQ ID NO;
21);F4;
QNAHRKT (SEQ ID NO;
22);F5;
RSDHLSE (SEQ ID NO;
48); andF6;
TSSTRKT (SEQ ID NO;
59);(x) F1;
TSGNLTR(SEQ ID NO;
60);F2;
QSGNLAR (SEQ ID No;
61);F3;
RSDALSV (SEQ ID NO;
62);F4;
DSSHRTR (SEQ ID NO;
63); andF5;
RSDVLSE (SEQ ID NO;
64)(xi) F1;
RSDNLSE (SEQ ID NO;
21);F2;
QNAHRKT (SEQ ID NO;
22);F3;
RSDHLSE (SEQ ID NO;
48);F4;
TSSTRKT (SEQ ID NO;
59); andF5;
TSGHLSR (SEQ ID NO;
40);(xii) F1;
TSGNLTR (SEQ ID NO;
60);F2;
QSGNLAR (SEQ ID NO;
61);F3;
RSDALSV (SEQ ID NO;
62); andF4;
DSSHRTR (SEQ ID NO;
63);(xiii) F1;
QRSNLVR (SEQ ID NO;
65);F2;
RSDHLTQ (SEQ ID NO;
66);F3;
QSGHLQR (SEQ ID NO;
67); andF4;
DRSHLAR (SEQ ID NO;
68);(xiv) F1;
RSDVLSE (SEQ ID NO;
64);F2;
QRNHRTT (SEQ ID NO;
69);F3;
TKRSLIE (SEQ ID NO;
70);F4;
TSSNLSR (SEQ ID NO;
71);F5;
RSDDLSK (SEQ ID NO;
25); andF6;
DNRDRIK (SEQ ID NO;
55);(xv) F1;
RSDHLSA (SEQ ID NO;
72);F2;
QSGHLSR (SEQ ID NO;
24);F3;
RSDHLSR (SEQ ID NO;
47);F4;
QNDNRIK (SEQ ID NO;
73); andF5;
QSGNLAR (SEQ ID NO;
61);(xvi) F1;
NNRDLIN (SEQ ID NO;
74);F2;
TSSNLSR (SEQ ID NO;
71);F3;
RSDVLSE (SEQ ID NO;
64);F4;
QRNHRTT (SEQ ID NO;
69);F5;
TKRSLIE (SEQ ID NO;
70); andF6;
TSSNLSR (SEQ ID NO;
71). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification