Patent ID: 11912985
Assignee: THE BROAD INSTITUTE, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.