US 11,912,985 B2
Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
David R. Liu, Cambridge, MA (US); Andrew Vito Anzalone, Cambridge, MA (US); Jonathan Ma Levy, Cambridge, MA (US); Xin Gao, Cambridge, MA (US); and Christopher J. Podracky, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Nov. 7, 2022, as Appl. No. 18/053,269.
Application 18/053,269 is a continuation of application No. PCT/US2021/031439, filed on May 7, 2021.
Claims priority of provisional application 63/116,785, filed on Nov. 20, 2020.
Claims priority of provisional application 63/022,397, filed on May 8, 2020.
Prior Publication US 2023/0220374 A1, Jul. 13, 2023
Int. Cl. C12N 15/10 (2006.01); C12N 9/12 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 9/1276 (2013.01); C12N 9/22 (2013.01); C12N 15/111 (2013.01)] 29 Claims
 
1. A system for simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited, said system comprising:
(a) a prime editor or one or more polynucleotides encoding the prime editor, wherein the prime editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a polypeptide comprising an RNA-dependent DNA polymerase activity, wherein the napDNAbp comprises a RuvC nuclease domain and a HNH nuclease domain, and wherein the HNH nuclease domain comprises one or more mutations that decrease or eliminate its nuclease activity;
(b) a first prime editing guide RNA (first PEgRNA) or one or more polynucleotides encoding the first PEgRNA, wherein the first PEgRNA comprises
(i) a first spacer sequence that is complementary to a first binding site on a first strand of the double-stranded DNA sequence upstream of the target site relative to the second strand,
(ii) a first gRNA core that is capable of complexing with the prime editor,
(iii) a first DNA synthesis template that encodes a first single-stranded DNA sequence, and
(iv) a first primer binding site complementary to a region of the second strand upstream of a first cut site; and
(c) a second prime editing guide RNA (second PEgRNA) or one or more polynucleotides encoding the second PEgRNA, wherein the second PEgRNA comprises
(i) a second spacer sequence that is complementary to a second binding site on a second strand of the double-stranded DNA sequence downstream of the target site relative to the second strand;
(ii) a second gRNA core that is capable of complexing with the prime editor,
(iii) a second DNA synthesis template that encodes a second single-stranded DNA sequence, and
(iv) a second primer binding site complementary to a region of the first strand upstream of a second cut site;
wherein the prime editor is capable of cleaving the first strand at the a-first cut site when complexed with the second PEgRNA and cleaving the second strand at the a-second cut site when complexed with the first PEgRNA,
wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence are reverse complements over a region of complementarity of each single-stranded DNA sequence of
at least 5 nucleotides in length, and
wherein the first single-stranded DNA sequence comprises a first edit compared to the second strand of the target site that starts at a position no more than 3 nucleotides from the first cut site.