Uses of adenosine base editors
The disclosure provides methods and compositions for treating blood diseases/disorders, such as sickle cell disease, hemochromatosis, hemophilia, and beta-thalassemia. For example the disclosure provides therapeutic guide RNAs that target the promotor of HBG1/2 to generate point mutations that increase expression of fetal hemoglobin. As another example, the disclosure provides therapeutic guide RNAs that target mutations in HBB, Factor VIII, and HFE to treat sickle cell disease, beta-thalassemia, hemophilia and hemochromatosis. The disclosure also provides fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA. In some embodiments, the fusion proteins are in complex with nucleic acids, such as guide RNAs (gRNAs), which target the fusion proteins to a DNA sequence (e.g., an HBG1 or HBG2 protmoter sequence, or an HFE, GBB, or F8 gene sequence). Such complexes may be useful for increasing expression of fetal hemoglobin or correcting a poing mutation (e.g., C282Y) in HFE.
1. A method for deaminating an adenosine (A) nucleobase in a sense or antisense strand of a promoter of an HBG1 or HBG2 gene, the method comprising contacting the promoter with a base editor and a guide RNA bound to the base editor, wherein the guide RNA (gRNA) comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter of at least one of the HBG1 and HBG2 gene, wherein the base editor comprises a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain, and (ii) an adenosine deaminase, wherein the adenosine deaminase comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of a wild-type adenosine deaminase, with the exception of substitution(s) at one or more of W23, H36, N37, P48, I49, R51, N72, L84, S97, A106, D108, H123, G125, A142, S146, D147, R152, E155, I156, K157, and K161 of the amino acid sequence of SEQ ID NO: 1, or corresponding substitution(s) in another wild-type adenosine deaminase, wherein the step of contacting the promoter results in a T-A base pair in the promoter being mutated to a C-G base pair in the promoter.
2. The method of claim 1 , wherein the target nucleic acid sequence comprises:
(SEQ ID NO: 838)
5′-CTTGGGGGCCCCT T CCCCACACTA-3′;
(SEQ ID NO: 839)
5′-CTTGGGGGCCCCT T CCCCACACT-3′;
(SEQ ID NO: 840)
5′-CTTGGGGGCCCCT T CCCCACAC-3′;
(SEQ ID NO: 841)
5′-CTTGGGGGCCCCT T CCCCACA-3′;
(SEQ ID NO: 842)
5′-CTTGGGGGCCCCT T CCCCAC-3′;
(SEQ ID NO: 843)
5′-CTTGGGGGCCCCT T CCCCA-3′;
(SEQ ID NO: 844)
5′-CTTGGGGGCCCCT T CCCC-3′;
[[or]]
(SEQ ID NO: 845)
5′-CTTGGGGGCCCCT T CCC-3′;
(SEQ ID NO: 297)
5′-CCACACTATCTCAATGCAAA T ATCTGTC-3′;
(SEQ ID NO: 298)
5′-CCACACTATCTCAATGCAAA T ATCTGT-3′;
(SEQ ID NO: 299)
5′-CCACACTATCTCAATGCAAA T ATCTG-3′;
(SEQ ID NO: 300)
5′-CCACACTATCTCAATGCAAA T ATCT-3′;
(SEQ ID NO: 301)
5′-CCACACTATCTCAATGCAAA T ATC-3′;
(SEQ ID NO: 302)
5′-CCACACTATCTCAATGCAAA T AT-3′;
(SEQ ID NO: 303)
5′-CCGTTTCAGACAGA T A TTT GCAT-3′;
(SEQ ID NO: 304)
5′-CCAGGGACCGTTTCAGACAGA T ATTTGC-3′;
(SEQ ID NO: 305)
5′-CCAGGGACCGTTTCAGACAGA T ATTTG-3′;
(SEQ ID NO: 306)
5′-CCAGGGACCGTTTCAGACAGA T ATTT-3′;
(SEQ ID NO: 307)
5′-CCAGGGACCGTTTCAGACAGA T ATT-3′;
(SEQ ID NO: 308)
5′-CCAGGGACCGTTTCAGACAGA T AT-3′;
(SEQ ID NO: 309)
5′-CCAGGGACCGTTTCAGACAGA T A-3′;
(SEQ ID NO: 310)
5′-ACACTATCTCAATGCAAA T ATCT-3′;
(SEQ ID NO: 311)
5′-ACTATCTCAATGCAAA T ATCTGT-3′;
(SEQ ID NO: 312)
5′-CCTCTTGGGGGCCCC TT CCCCAC-3′;
(SEQ ID NO: 313)
5′-CCTTGTCAAGGCTA TT GG T CAAG-3′;
(SEQ ID NO: 314)
5′-CCTTGCCTTGACCAA T AGCC TT GACAAG-3′;
(SEQ ID NO: 315)
5′-CCTTGCCTTGACCAA T AGCC TT GACAA-3′;
(SEQ ID NO: 316)
5′-CCTTGCCTTGACCAA T AGCC TT GACA-3′;
(SEQ ID NO: 317)
5′-CCTTGCCTTGACCAA T AGCC TT GAC-3′;
(SEQ ID NO: 318)
5′-CCTTGCCTTGACCAA T AGCC TT GA-3′;
(SEQ ID NO: 319)
5′-CCTTGCCTTGACCAA T AGCC TT G-3′;
(SEQ ID NO: 320)
5′-GCCTTGACCAATAGCC TT GACAA-3′;
(SEQ ID NO: 321)
5′-CCTTGACCAATAGCC TT GACAAG-3′;
(SEQ ID NO: 322)
5′-GCCTTGTCAAGGCTA TT GG T CAA-3′;
or
(SEQ ID NO: 323)
5′-TCAAGTTTGCCTTG T CAAGGCTA-3′.
3. The method of claim 1 , wherein the guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 846)
5′-UCAUGUGGGG A AGGGGCCCCCAAG-3′;
(SEQ ID NO: 847)
5′-CAUGUGGGG A AGGGGCCCCCAAG-3′;
(SEQ ID NO: 848)
5′-AUGUGGGG A AGGGGCCCCCAAG-3′;
(SEQ ID NO: 849)
5′-UGUGGGG A AGGGGCCCCCAAG-3′.
(SEQ ID NO: 850)
5′-GUGGGG A AGGGGCCCCCAAG-3′;
(SEQ ID NO: 851)
5′-UGGGG A AGGGGCCCCCAAG-3′;
(SEQ ID NO: 852)
5′-GGGG A AGGGGCCCCCAAG-3′;
or
(SEQ ID NO: 853)
5′-GGG A AGGGGCCCCCAAG-3′.
4. The method of claim 1 , wherein the target nucleic acid sequence comprises:
(SEQ ID NO: 838)
5′-CTTGGGGGCCCCT T CCCCACACTA-3′;
(SEQ ID NO: 839)
5′-CTTGGGGGCCCCT T CCCCACACT-3′;
(SEQ ID NO: 840)
5′-CTTGGGGGCCCCT T CCCCACAC-3′;
(SEQ ID NO: 841)
5′-CTTGGGGGCCCCT T CCCCACA-3′;
(SEQ ID NO: 842)
5′-CTTGGGGGCCCCT T CCCCAC-3′;
(SEQ ID NO: 843)
5′-CTTGGGGGCCCCT T CCCCA-3′;
(SEQ ID NO: 844)
5′-CTTGGGGGCCCCT T CCCC-3′;
or
(SEQ ID NO: 845)
5′-CTTGGGGGCCCCT T CCC-3′;
and
wherein deamination of the A nucleobase that is complementary to the T at position 14 of SEQ ID NO: 845 results in a T to C mutation in the target nucleic acid sequence.
5. The method of claim 1 , wherein the guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 254)
5′-GACAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 255)
5′-ACAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 256)
5′-CAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 257)
5′-AGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 258)
5′-GAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 259)
5′-AU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 260)
5′-AUGCAAAUAUCUGUCUGAAACGG-3′;
(SEQ ID NO: 261)
5′-GCAAAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 262)
5′-CAAAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 263)
5′-AAAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 264)
5′-AAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 265)
5′-AU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 266)
5′-U A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 267)
5′-AGAU A UUUGCAUUGAGAUAGUGU-3′;
(SEQ ID NO: 268)
5′-ACAGAU A UUUGCAUUGAGAUAGU-3′;
(SEQ ID NO: 269)
5′-GUGGGG AA GGGGCCCCCAAGAGG-3′;
(SEQ ID NO: 270)
5′-CUUG A CC AA UAGCCUUGACAAGG-3′;
(SEQ ID NO: 271)
5′-CUUGUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 272)
5′-UUGUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 273)
5′-UGUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 274)
5′-GUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 275)
5′-UC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 276)
5′-C AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 277)
5′-UUGUC AA GGCUAUUGGUCAAGGC-3′;
(SEQ ID NO: 278)
5′-CUUGUC AA GGCUAUUGGUCAAGG-3′;
(SEQ ID NO: 279)
5′-UUG A CC AA UAGCCUUGACAAGGC-3′;
(SEQ ID NO: 280)
5′-UAGCCUUG A CAAGGCAAACUUGA-3′.
6. The method of claim 5 , wherein the target nucleic acid sequence comprises
(SEQ ID NO: 297)
5′-CCACACTATCTCAATGCAAA T ATCTGTC-3′;
(SEQ ID NO: 298)
5′-CCACACTATCTCAATGCAAA T ATCTGT-3′;
(SEQ ID NO: 299)
5′-CCACACTATCTCAATGCAAA T ATCTG-3′;
(SEQ ID NO: 300)
5′-CCACACTATCTCAATGCAAA T ATCT-3′;
(SEQ ID NO: 301)
5′-CCACACTATCTCAATGCAAA T ATC-3′;
or
(SEQ ID NO: 302)
5′-CCACACTATCTCAATGCAAA T AT-3′;
and wherein deamination of the A nucleobase that is complementary to the T at position 21 of SEQ ID NO: 302 results in a T to C mutation in the target nucleic acid sequence.
7. The method of claim 5 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein:
(a) the second target nucleic acid sequence comprises SEQ ID NO: 845;
(b) the second guide sequence comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 853; or
(c) both (a) and (b).
8. The method of claim 1 , wherein the guide sequence of the gRNA comprises at least 15 contiguous nucleotides of the sequence
(SEQ ID NO: 270)
5′-CUUG A CC AA UAGCCUUGACAAGG-3′.
9. The method of claim 8 , wherein the target nucleic acid sequence comprises 5′-CCTTGTCAAGGCTATTGGTCAAG-3′ (SEQ ID NO: 313);
wherein deamination of an A nucleobase that is complementary to the T at any one of positions 15, 16, or 19 of SEQ ID NO: 313 results in a T to C mutation in the target nucleic acid sequence.
10. The method of claim 8 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein:
(a) the second target nucleic acid sequence comprises SEQ ID NO: 845, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 309, SEQ ID NO: 311, or SEQ ID NO: 312;
(b) the second guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 853, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 268, and SEQ ID NO: 269; or
(c) both (a) and (b).
11. The method of claim 1 further comprising deaminating a second A nucleobase in the sense or antisense strand of the promoter using a second gRNA comprising a second guide sequence that is complementary to a second target nucleic acid sequence, wherein:
(a) the second target nucleic acid sequence comprises SEQ ID NO: 845, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 319, SEQ ID NO: 320, or SEQ ID NO: 321;
(b) the second guide sequence comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 853, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278; or
(c) both (a) and (b).
12. The method of claim 1 , wherein the step of contacting the promoter leads to an increase in transcription of the HBG1 gene or the HBG2 gene.
13. The method of claim 1 , wherein the method is performed in a cell of a subject who has a disease or disorder of the blood.
14. The method of claim 13 , wherein the disease or disorder is caused by a mutation in a gene, or a promoter of a gene, selected from the group consisting of CYGB, HBA1, HBA2, HBB, HBD, HBE1, HBG1, HBG2, HBM, HBQ1, HBZ, and MB.
15. The method of claim 1 , wherein the adenosine deaminase comprises each of the W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N substitutions in SEQ ID NO: 1, or the corresponding substitutions in another wild-type adenosine deaminase.
16. The method of claim 1 , wherein the guide sequence of the guide RNA comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 254)
5′-GACAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 255)
5′-ACAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 256)
5′-CAGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 257)
5′-AGAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 258)
5′-GAU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 259)
5′-AU A UUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO: 260)
5′-AUGC AAA U A UCUGUCUGAAACGG-3′;
(SEQ ID NO: 261)
5′-GCAAAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 262)
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 263)
5′-AAAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 264)
5′-AAU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 265)
5′-AU A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 266)
5′-U A UCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO: 267)
5′-AGAU A UUUGCAUUGAGAUAGUGU-3′;
(SEQ ID NO: 268)
5′-ACAGAU A UUUGCAUUGAGAUAGU-3′;
(SEQ ID NO: 269)
5′-GUGGGG AA GGGGCCCCCAAGAGG-3′;
(SEQ ID NO: 270)
5′-CUUG A CC AA UAGCCUUGACAAGG-3′;
(SEQ ID NO: 271)
5′-CUUGUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 272)
5′-UUGUC AA GGCU A UUGGUCAAGGCAAGG-3′ ;
(SEQ ID NO: 273)
5′-UGUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 274)
5′-GUC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 275)
5′-UC AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 276)
5′-C AA GGCU A UUGGUCAAGGCAAGG-3′;
(SEQ ID NO: 277)
5′-UUGUC AA GGCUAUUGGUCAAGGC-3′;
(SEQ ID NO: 278)
5′-CUUGUC AA GGCUAUUGGUCAAGG-3′;
(SEQ ID NO: 279)
5′-UUG A CC AA UAGCCUUGACAAGGC-3′;
or
(SEQ ID NO: 280)
5′-UAGCCUUG A CAAGGCAAACUUGA-3′.
17. The method of claim 1 , wherein the method causes less than 1% indel formation.
18. The method of claim 1 , wherein the efficiency of deaminating an A nucleobase is at least 30%.
19. The method of claim 1 , wherein the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to SEQ ID NO: 270.
20. The method of claim 19 , wherein the nucleobase changes comprise a deletion of one or more nucleobases of SEQ ID NO: 270.
21. The method of claim 20 , wherein the gRNA comprises the nucleic acid sequence 5′-CUUGACCAAUAGCCUUGACA-3′ (corresponding to bases 1-20 of SEQ ID NO: 270).
22. The method of claim 20 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
23. The method of claim 1 , wherein the guide sequence of the gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase changes relative to SEQ ID NO: 260.
24. The method of claim 19 , wherein the nucleobase changes comprise a deletion of one or more nucleobases of SEQ ID NO: 260.
25. The method of claim 24 , wherein the gRNA comprises the nucleic acid sequence 5′-AUGCAAAUAUCUGUCUGAAA-3′ (corresponding to bases 1-20 of SEQ ID NO: 260).
26. The method of claim 24 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
27. The method of claim 2 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
28. The method of claim 4 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
29. The method of claim 6 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
30. The method of claim 7 , wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
31. The method of claim 9 , wherein the target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
32. The method of claim 10 , wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
33. The method of claim 11 , wherein the second target nucleic acid sequence comprises a 3′ end that is not immediately adjacent to a protospacer adjacent motif (PAM) site.
34. The method of claim 1 , wherein the adenosine deaminase comprises one or more of W23L, W23R, H36L, N37S, P48S, P48A, I49V, R51L, N72D, L84F, S97C, A106V, D108N, H123Y, G125A, A142N, S146C, D147Y, R152P, E155V, I156F, K157N, and/or K161T substitutions in SEQ ID NO: 1, or one or more corresponding substitutions in another wild-type adenosine deaminase.
35. The method of claim 1 , wherein the adenosine deaminase comprises one or more of W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and/or K157N substitutions in SEQ ID NO: 1, or one or more corresponding substitutions in another wild-type adenosine deaminase.
36. The method of claim 1 , wherein the adenosine deaminase comprises A106V and D108N substitutions in SEQ ID NO: 1, or corresponding substitutions in another wild-type adenosine deaminase.
37. The method of claim 1 , wherein the adenosine deaminase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 65-83, 420-437, 672-684, and 802-805.
38. The method of claim 1 , wherein the adenosine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 65-83, 420-437, 672-684, and 802-805.
39. The method of claim 1 , wherein the napDNAbp domain comprises the amino acid sequence of any one of SEQ ID NOs: 34-36 or 417-419.