HOMING ENDONUCLEASE VARIANTS
The present disclosure provides homing endonuclease variants and megaTALs reprogrammed to bind and cleave a polynucleotide sequence in the genome. The homing endonuclease variants and megaTALs have been engineered to increase the thermostability and/or activity.
1 . An I-OnuI homing endonuclease (HE) variant comprising one or more amino acid substitutions relative to a parent I-OnuI HE comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
2 . The I-OnuI HE variant of claim 1 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, V116, F168, D208, N246, and L263.
3 . The I-OnuI HE variant of claim 1 , wherein the I-OnuI HE variant comprises amino acid substitutions at the following amino acid positions: I14, A19, F168, D208, and N246.
4 . The I-OnuI HE variant of claim 1 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: K108, K156, S176, E231, V261, E277, and G300.
5 . The I-OnuI HE variant of claim 1 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
6 . The I-OnuI HE variant of claim 1 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
7 . The I-OnuI HE variant of claim 1 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
8 . The I-OnuI HE variant of claim 1 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
9 . The I-OnuI HE variant of claim 1 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
10 . The I-OnuI HE variant of any one of claims 1 to 9 , wherein the I-OnuI HE variant has a TM 50 at least 10° C. higher than the TM 50 of the parent I-OnuI HE.
11 . The I-OnuI HE variant of any one of claims 1 to 9 , wherein the I-OnuI HE variant has a TM 50 at least 15° C. higher than the TM 50 of the parent I-OnuI HE.
12 . The I-OnuI HE variant of any one of claims 1 to 9 , wherein the I-OnuI HE variant has a TM 50 at least 20° C. higher than the TM 50 of the parent I-OnuI HE.
13 . The I-OnuI HE variant of any one of claims 1 to 9 , wherein the I-OnuI HE variant has a TM 50 at least 25° C. higher than the TM 50 of the parent I-OnuI HE.
14 . The I-OnuI HE variant of any one of claims 1 to 13 , wherein the I-OnuI HE variant targets a site in a gene selected from the group consisting of: HBA, HBB, HBG1, HBG2, BCL11A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CIITA, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Ra2, TRAILR1, RCASIR, and FAS.
15 . An I-OnuI homing endonuclease (HE) variant comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
16 . The I-OnuI HE variant of claim 15 , wherein the parent I-OnuI HE amino acid sequence set is forth in SEQ ID NO: 1.
17 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, V116, F168, D208, N246, and L263.
18 . The I-OnuI HE variant of any one of claims 15 to 17 , wherein the I-OnuI HE variant comprises amino acid substitutions at the following amino acid positions: I14, A19, F168, D208, and N246.
19 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for I14 is selected from the group consisting of: S, N, M, K, F, D, T, and V.
20 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for I14 is selected from the group consisting of: T and V.
21 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for A19 is selected from the group consisting of: C, D, I, L, S, T and V.
22 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for A19 is selected from the group consisting of: T and V.
23 . The I-OnuI HE variant of claim 17 , wherein the amino acid substituted for V116 is selected from the group consisting of: F, D, A, L and I.
24 . The I-OnuI HE variant of claim 17 , wherein the amino acid substituted for V116 is selected from the group consisting of: L and I.
25 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for F168 is selected from the group consisting of: H, Y, I, V, P, L and S.
26 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for F168 is selected from the group consisting of: L and S.
27 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for D208 is selected from the group consisting of: N, V, Y, and E.
28 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for D208 is E.
29 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for N246 is selected from the group consisting of: H, I, D, R, S, T, V, Y, and K.
30 . The I-OnuI HE variant of claim 17 or claim 18 , wherein the amino acid substituted for N246 is K.
31 . The I-OnuI HE variant of claim 17 , wherein the amino acid substituted for L263 is selected from the group consisting of: H, F, P, T, V, and R.
32 . The I-OnuI HE variant of claim 17 , wherein the amino acid substituted for L263 is R.
33 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: K108, K156, S176, E231, V261, E277, and G300.
34 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for K108 is selected from the group consisting of: E, N, Q, R, T, V, and M.
35 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for K108 is M
36 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for K156 is selected from the group consisting of: N, Q, R, T, V, I, and E.
37 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for K156 is selected from the group consisting of: I and E
38 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for S176 is selected from the group consisting of: P, N and A.
39 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for S176 is A.
40 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for E231 is selected from the group consisting of: D, K, V, and G.
41 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for E231 is selected from the group consisting of: K and G.
42 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for V261 is selected from the group consisting of: D, G, I, L, S, T, and A.
43 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for V261 is A.
44 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for E277 is selected from the group consisting of: A, D, G, Q, V, and K.
45 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for E277 is K.
46 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for G300 is selected from the group consisting of: S, V, D, C, and R.
47 . The I-OnuI HE variant of claim 33 , wherein the amino acid substituted for G300 is R.
48 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
49 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for N31 is selected from the group consisting of: D, H, I, R, K, S, T and Y.
50 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for N31 is K.
51 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for N33 is selected from the group consisting of: D, G, H, I, K, S, T and Y.
52 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for N33 is K.
53 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K52 is selected from the group consisting of: Q, R, T, Y, N, E, and M.
54 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K52 is M.
55 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for Y97 is selected from the group consisting of: F, N and H.
56 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for Y97 is F.
57 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K124 is selected from the group consisting of: E, N, R and T.
58 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K124 is N.
59 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K147 is selected from the group consisting of: E, I, N, R and T.
60 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K147 is I.
61 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for 1153 is selected from the group consisting of: D, H, K, T, Y, S, V and N.
62 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for 1153 is N.
63 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K209 is selected from the group consisting of: E, M, N, Q and R.
64 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for K209 is R.
65 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for E264 is selected from the group consisting of: A, D, G, K, Q, R and V.
66 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for E264 is K.
67 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for D268 is selected from the group consisting of: A, E, G, H, N, V and Y.
68 . The I-OnuI HE variant of claim 48 , wherein the amino acid substituted for D268 is N.
69 . The I-OnuI HE variant of any one of claims 15 to 68 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions.
70 . The I-OnuI HE variant of any one of claims 15 to 68 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions.
71 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
72 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
73 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
74 . The I-OnuI HE variant of claim 15 or claim 16 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
75 . The I-OnuI HE variant of any one of claims 15 to 74 , wherein the I-OnuI HE variant has a TM 50 at least 10° C. higher than the TM 50 of the parent I-OnuI HIE.
76 . The I-OnuI HE variant of any one of claims 15 to 74 , wherein the I-OnuI HE variant has a TM 50 at least 15° C. higher than the TM 50 of the parent I-OnuI HE.
77 . The I-OnuI HE variant of any one of claims 15 to 74 , wherein the I-OnuI HE variant has a TM 50 at least 20° C. higher than the TM 50 of the parent I-OnuI HE.
78 . The I-OnuI HE variant of any one of claims 15 to 74 , wherein the I-OnuI HE variant has a TM 50 at least 25° C. higher than the TM 50 of the parent I-OnuI HE.
79 . The I-OnuI HE variant of any one of claims 15 to 78 , wherein the I-OnuI HE variant targets a site in a gene selected from the group consisting of: HBA, HBB, HBG1, HBG2, BCL11A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CIITA, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Ra2, TRAILR1, RCASIR, and FAS.
80 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human BCL11A gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
81 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human PCSK9 gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
82 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human PDCD-1 gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
83 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human TCRα gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
84 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human CBLB gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
85 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human CTLA-4 gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
86 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human TGFBRII gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
87 . An I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human TIM3 gene, comprising one or more amino acid substitutions relative to a parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermostability of the I-OnuI HE variant compared to the parent I-OnuI HE.
88 . The I-OnuI HE variant of any one of claims 80 to 87 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
89 . The I-OnuI HE variant of any one of claims 80 to 87 , wherein the I-OnuI HE variant comprises amino acid substitutions at the following amino acid positions: I14, A19, F168, D208, and N246.
90 . The I-OnuI HE variant of any one of claims 80 to 87 , wherein the I-OnuI HE variant comprises three or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
91 . The I-OnuI HE variant of any one of claims 80 to 87 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300.
92 . The I-OnuI HE variant of any one of claims 80 to 87 , wherein the I-OnuI HE variant comprises five or more amino acid substitutions is at an amino acid position selected from the group consisting of: I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300; and one or more amino acid substitutions is at an amino acid position selected from the group consisting of: N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268.
93 . The I-OnuI HE variant of any one of claims 80 to 92 , wherein the I-OnuI HE variant has a TM 50 at least 10° C. higher than the TM 50 of the parent I-OnuI HE.
94 . The I-OnuI HE variant of any one of claims 80 to 92 , wherein the I-OnuI HE variant has a TM 50 at least 15° C. higher than the TM 50 of the parent I-OnuI HE.
95 . The I-OnuI HE variant of any one of claims 80 to 92 , wherein the I-OnuI HE variant has a TM 50 at least 20° C. higher than the TM 50 of the parent I-OnuI HE.
96 . The I-OnuI HE variant of any one of claims 80 to 92 , wherein the I-OnuI HE variant has a TM 50 at least 25° C. higher than the TM 50 of the parent I-OnuI HE.