METHODS AND COMPOSITIONS FOR SIMULTANEOUS EDITING OF BOTH STRANDS OF A TARGET DOUBLE-STRANDED NUCLEOTIDE SEQUENCE

1 - 232 . (canceled)

233 . 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 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 a second strand of the double-stranded DNA sequence,

(ii) a first gRNA core, and

(iii) a first RNA extension arm comprising a first DNA synthesis template and a first primer binding site; and

(b) 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 the second strand of the double-stranded DNA sequence downstream of the target site relative to the second strand,

(ii) a second gRNA core, and

(iii) a second RNA extension arm comprising a second DNA synthesis template and a second primer binding site;

wherein the first gRNA core and the second gRNA core are each capable of complexing with a nucleic acid programmable DNA binding protein (napDNAbp) that has cleavage activity to cleave the second strand at a first cut site when complexed with the first PEgRNA and cleave the first strand at a second cut site when complexed with the second PEgRNA;

wherein the first DNA synthesis template encodes a first single-stranded DNA sequence;

wherein the second DNA synthesis template encodes a second single-stranded DNA sequence;

wherein the first primer binding site is complementary to a region of the second strand upstream of the first cut site;

wherein the second primer binding site is complementary to a region of the first strand upstream of the second cut site;

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; 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.

234 . The system of claim 233 , wherein the second single-stranded DNA sequence comprises a second edit compared to the first strand of the target site that starts at a position no more than 3 nucleotides from the second cut site.

235 . The system of claim 233 , wherein the first edit starts at a position no more than 2 nucleotides from the first cut site.

236 . The system of claim 233 , wherein the first edit starts at the first cut site.

237 . The system of claim 234 , wherein the second edit starts at a position no more than 2 nucleotides from the second cut site.

238 . The system of claim 234 , wherein the second edit starts at the second cut site.

239 . The system of claim 233 , wherein the region of complementarity encompasses the 3′ ends of the first and the second single-stranded DNA sequences.

240 . The system of claim 233 , wherein the region of complementarity is at least 10 nucleotides in length.

241 . The system of claim 233 , wherein the first single-stranded DNA sequence is the reverse complement of the second single-stranded DNA sequence.

242 . The system of claim 233 , further comprising a prime editor protein, or one or more polynucleotides encoding the prime editor protein, wherein the prime editor protein comprises a napDNAbp and an RNA-dependent DNA polymerase, wherein the napDNAbp comprises a RuvC nuclease domain that is capable of cleaving the second strand at the first cut site when complexed with the first PEgRNA and cleaving the first strand at the second cut site when complexed with the second PEgRNA.

243 . The system of claim 242 , wherein the napDNAbp comprises a HNH nuclease domain.

244 . The system of claim 242 , wherein the napDNAbp is a Cas9.

245 . The system of claim 242 , wherein the first single-stranded DNA sequence, the second single-stranded sequence, or the region of complementarity comprises a recombinase recognition sequence.

246 . The system of claim 245 , further comprising a recombinase capable of recognizing the recombinase recognition sequence, or one or more polynucleotides encoding the recombinase.

247 . The system of claim 246 , wherein the recombinase is Bxb1, and wherein the recombinase recognition sequence comprises SEQ ID NO: 536 or SEQ ID NO: 537.

248 . The system of claim 245 , further comprising a donor template or a polynucleotide encoding the donor template.

249 . The system of claim 248 , wherein the recombinase recognition sequence comprises SEQ ID NO: 536, and the donor template comprises SEQ ID NO: 537, or wherein the recombinase recognition sequence comprises SEQ ID NO: 537, and the donor template comprises SEQ ID NO: 536.

250 . The system of claim 248 , wherein the target site is in intron 1 of a target gene.

251 . The system of claim 248 , wherein the donor template comprises a cDNA sequence of the target gene and a splice acceptor sequence 5′ to the cDNA sequence.

252 . The system of claim 242 , wherein the RNA-dependent DNA polymerase comprises an amino acid sequence of any one of SEQ ID NOs: 89-100, 106-122, 128, 132, 139, 143, 149, 154, 159, 700-736, 738-742, and 763-766, or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 89-100, 106-122, 128, 132, 139, 143, 149, 154, 159, 700-736, 738-742, and 763-766.