Methods and compositions for prime editing nucleotide sequences
Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.
1. A prime editing guide RNA (PEgRNA) comprising:
a spacer sequence that comprises a region of complementarity to a first strand of a double-stranded target human DNA sequence,
an RNA extension arm that comprises a DNA synthesis template and a primer binding site in a 5′ to 3′ orientation, and
a gRNA core that associates with a nucleic acid programmable DNA binding protein (napDNAbp) having nicking activity,
wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in a second strand of the double-stranded target DNA sequence;
wherein the DNA synthesis template comprises a region of complementarity to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence, and comprises one or more nucleotide changes compared to the double-stranded target DNA sequence;
wherein the napDNAbp is a Cas9, a Cas12a, or a Cas12b1; and
wherein the first strand and second strand of the double-stranded target DNA sequence comprise a region of complementarity to each other.
2. The PEgRNA of claim 1 , wherein the spacer, the RNA extension arm, and the gRNA core are in a single RNA molecule.
3. The PEgRNA of claim 2 , comprising in a 5′ to 3′ orientation: the spacer, the gRNA core, the DNA synthesis template, and the primer binding site.
4. The PEgRNA of claim 2 , comprising in a 5′ to 3′ orientation: the DNA synthesis template, the primer binding site, the spacer, and the gRNA core.
5. The PEgRNA of claim 2 , wherein the extension arm is flanked by a 5′ fragment of the gRNA core and a 3′ fragment of the gRNA core.
6. The PEgRNA of claim 1 , wherein the DNA synthesis template is from 3 nucleotides to 500 nucleotides in length.
7. The PEgRNA of claim 6 , wherein the DNA synthesis template is from 5 to 58 nucleotides in length.
8. The PEgRNA of claim 6 , wherein the DNA synthesis template is from 8 to 31 nucleotides in length.
9. The PEgRNA of claim 1 , wherein the one or more nucleotide changes comprises an insertion of one or more nucleotides as compared to the double-stranded target DNA sequence.
10. The PEgRNA of claim 1 , wherein the one or more nucleotide changes comprises one or more nucleotide substitutions as compared to the double-stranded target DNA sequence.
11. The PEgRNA of claim 1 , wherein the one or more nucleotide changes comprises a deletion of one or more nucleotides as compared to the double-stranded target DNA sequence.
12. The PEgRNA of claim 1 , wherein the one or more nucleotide changes comprises insertions of one or more nucleotides, one or more nucleotide substitutions, deletions of one or more nucleotides, or a combination of any such nucleotide changes, as compared to the double-stranded target DNA sequence.
13. The PEgRNA of claim 1 , wherein the DNA synthesis template and the primer binding site are directly adjacent to each other.
14. The PEgRNA of claim 1 , wherein the primer binding site is from 3 nucleotides to 500 nucleotides in length.
15. The PEgRNA of claim 14 , wherein the primer binding site is from 3 to 20 nucleotides in length.
16. The PEgRNA of claim 14 , wherein the primer binding site is from 7 to 17 nucleotides in length.
17. The PEgRNA of claim 1 , wherein the DNA synthesis template is from 5 nucleotides to 58 nucleotides in length.
18. The PEgRNA of claim 1 , comprising at least one of: a chemically or biologically modified nucleobase, a modified sugar, a modified phosphate group, or a nucleoside analog.
19. The PEgRNA of claim 1 , wherein the DNA synthesis template is from 8 nucleotides to 31 nucleotides in length.
20. The PEgRNA of claim 1 , wherein the DNA synthesis template is from 10 nucleotides to 16 nucleotides in length.
21. The PEgRNA of claim 1 , wherein the DNA synthesis template is from 12 nucleotides to 17 nucleotides in length.
22. The PEgRNA of claim 1 , wherein the primer binding site is from 8 nucleotides to 15 nucleotides in length.
23. The PEgRNA of claim 1 , wherein the primer binding site is from 8 nucleotides to 11 nucleotides in length, and contains greater than about 60% GC content.
24. The PEgRNA of claim 1 , wherein at least one of the one or more nucleotide changes are at a position corresponding to a nucleotide within a protospacer adjacent motif (PAM) in the second strand of the double-stranded target DNA sequence.
25. The PEgRNA of claim 1 , wherein the napDNAbp is a Cas9.
26. The PEgRNA of claim 1 , wherein the nick site is three nucleotides upstream of a protospacer adjacent motif (PAM) in the second strand of the double-stranded target DNA sequence.
27. The PEgRNA of claim 1 , wherein the primer binding site comprises a region of complementarity to the spacer sequence.
28. The PEgRNA of claim 1 , wherein the spacer sequence is about 20 nucleotides in length.
29. The PEgRNA of claim 1 , wherein the primer binding site is the reverse complement of a region immediately 5′ of the nick site in the second strand.
30. The PEgRNA of claim 1 , wherein the spacer sequence is about 20 nucleotides in length.
31. The PEgRNA of claim 1 , wherein the one or more nucleotide changes is at a position corresponding to a nucleotide 1 to 6 bases downstream of the nick site in the second strand of the double-stranded target human DNA sequence, and wherein the DNA synthesis template is 17 nucleotides to 20 nucleotides in length.
32. The PEgRNA of claim 1 , wherein the one or more nucleotide changes is at a position corresponding to a nucleotide at least 7 bases downstream of the nick site in the second strand of the double-stranded target human DNA sequence, and wherein the DNA synthesis template is at least 12 nucleotides in length.
33. The PEgRNA of claim 1 , wherein the one or more nucleotide changes is a deletion or an insertion of at least 5 nucleotides, and wherein the DNA synthesis template comprises a sequence that is complementary to a sequence of at least 20 nucleotides downstream of a position of the deletion or insertion.
34. The PEgRNA of claim 1 , wherein the primer binding site is from 12 nucleotides to 13 nucleotides in length, and comprises about 40-60% G•C content.
35. The PEgRNA of claim 1 , wherein the primer binding site is from 14 nucleotides to 15 nucleotides in length, and contains less than about 40% GC content.
36. The PEgRNA of claim 1 , wherein the double stranded target human DNA is a disease-associated gene, and wherein the one or more nucleotide edits is capable of correcting a mutation in the disease-associated gene.
37. The PEgRNA of claim 1 , wherein:
A) the primer binding site is the reverse complement of a region immediately 5′ of the nick site in the second strand;
B) the primer binding site is from 8 to 15 nucleotides in length;
C) the DNA synthesis template is less than 15 nucleotides in length;
D) wherein at least one of the one or more nucleotide changes is at a position corresponding to a nucleotide within a protospacer adjacent motif (PAM) in the second strand of the double-stranded target human DNA sequence; or
E) wherein the 5′ most nucleotide of the RNA extension arm is not a cytosine;
or any combination thereof.
38. The PEgRNA of claim 37 , wherein the primer binding site comprises a region of complementarity to the spacer sequence.
39. The PEgRNA of claim 37 , wherein the nick site is three nucleotides upstream of a protospacer adjacent motif (PAM) in the second strand of the double-stranded target DNA sequence.
40. The PEgRNA of claim 37 , wherein the primer binding site is from 8 to 15 nucleotides in length.
41. The PEgRNA of claim 37 , wherein the DNA synthesis template is less than 15 nucleotides in length.
42. The PEgRNA of claim 37 , wherein the PEgRNA comprises, in a 5′ to 3′ orientation, the spacer, the gRNA core, the DNA synthesis template, and the primer binding site.
43. The PEgRNA of claim 37 , wherein the DNA synthesis template and the primer binding site are directly adjacent to each other.
44. The PEgRNA of claim 37 , wherein the napDNAbp is the Cas9.
45. The PEgRNA of claim 1 , wherein the primer binding site is from 3 to 20 nucleotides in length and is the reverse complement of a region immediately 5′ of the nick site, which is three nucleotides upstream of a protospacer adjacent motif (PAM) in the second strand of the double-stranded target DNA sequence.
46. The PEgRNA of claim 45 , wherein the primer binding site is from 7 to 17 nucleotides in length.
47. The PEgRNA of claim 45 , wherein the primer binding site is from 8 to 15 nucleotides in length.
48. The PEgRNA of claim 45 , wherein the DNA synthesis template is less than 15 nucleotides in length.
49. The PEgRNA of claim 45 , wherein the DNA synthesis template is from 5 nucleotides to 58 nucleotides in length.
50. The PEgRNA of claim 45 , wherein the DNA synthesis template is from 8 to 31 nucleotides in length.
51. The PEgRNA of claim 45 , comprising in a 5′ to 3′ orientation: the spacer, the gRNA core, the DNA synthesis template, and the primer binding site.
52. The PEgRNA of claim 45 , wherein the DNA synthesis template and the primer binding site are directly adjacent to each other.
53. The PEgRNA of claim 45 , wherein the spacer, the gRNA core, and the RNA extension arm are in a single RNA molecule.
54. The PEgRNA of claim 45 , wherein the napDNAbp is a Cas9.
55. The PEgRNA of claim 45 , wherein the 5′ most nucleotide of the RNA extension arm is not a cytosine.
56. The PEgRNA of claim 1 , wherein:
A) the PEgRNA comprises the spacer, the gRNA core, the DNA synthesis template, and the primer binding site in a 5′ to 3′ orientation in a single RNA molecule;
B) the primer binding site is the reverse complement of a region immediately 5′ of the nick site in the second strand;
C) the nick site is three nucleotides upstream of a protospacer adjacent motif (PAM) in the second strand of the double-stranded target DNA sequence;
D) the primer binding site is from 7 to 17 nucleotides in length;
E) the DNA synthesis template is from 5 nucleotides to 58 nucleotides in length; and
F) the DNA synthesis template and the primer binding site are directly adjacent to each other.
57. The PEgRNA of claim 56 , wherein at least one of the one or more nucleotide changes is at a position corresponding to a nucleotide within a protospacer adjacent motif (PAM) in the second strand of the double-stranded target human DNA sequence.
58. The PEgRNA of claim 56 , wherein the 5′ most nucleotide of the RNA extension arm is not a cytosine.
59. The PEgRNA of claim 56 , wherein the primer binding site is from 8 to 15 nucleotides in length.
60. The PEgRNA of claim 56 , wherein the DNA synthesis template is from 8 to 31 nucleotides in length.