EP4646482A2 - Genomeditierungszusammensetzungen und verfahren zur verwendung - Google Patents

Genomeditierungszusammensetzungen und verfahren zur verwendung

Info

Publication number
EP4646482A2
EP4646482A2 EP24739021.4A EP24739021A EP4646482A2 EP 4646482 A2 EP4646482 A2 EP 4646482A2 EP 24739021 A EP24739021 A EP 24739021A EP 4646482 A2 EP4646482 A2 EP 4646482A2
Authority
EP
European Patent Office
Prior art keywords
sequence
prime
seq
editing
editing system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24739021.4A
Other languages
English (en)
French (fr)
Inventor
Christopher J. PODRACKY
Emily POMEROY
Noah B. BLOCH
Andrew V. Anzalone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Prime Medicine Inc
Original Assignee
Prime Medicine Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Prime Medicine Inc filed Critical Prime Medicine Inc
Publication of EP4646482A2 publication Critical patent/EP4646482A2/de
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl

Definitions

  • Adoptive T cell therapy is an emerging cancer treatment modality.
  • Adoptive T cell therapy can involve the ex vivo manipulation of T cells to express T-cell receptors (TCRs) or chimeric antigen receptors (CARs) engineered to recognize a tumor specific antigen.
  • TCRs T-cell receptors
  • CARs chimeric antigen receptors
  • T cell therapy can involve autologous (i.e. patient-derived) T cells, which can avoid issues of immunogenicity and intolerance upon reintroduction of the ex vivo manipulated cells.
  • manufacturing challenges e.g., time, expense, poor quality/quantity
  • allogeneic (i.e., donor-derived) T cell immunotherapies make an attractive alternative.
  • An obstacle to allogeneic cell therapies is that the endogenous TCR present on the infused allogeneic T cells may recognize non-tumor antigens in the recipient, leading to graft-versus-host disease (GvHD).
  • the TCR contains a TCR ⁇ chain, encoded by a single T Cell Receptor Alpha Constant (TRAC) gene, complexed with a TCR ⁇ chain, encoded by two T Cell Receptor Beta Constant (TRBC) genes.
  • the TRAC gene is located in the human genome at 14q11.2.
  • TRAC mRNA is approximately 1.5 kb. Disruption of the endogenous TCR can be achieved by eliminating expression of the TCR ⁇ chain because the TCR ⁇ dimer is necessary for full function of TCR. Therefore, the alloreactive potential of donor T cells to elicit GvHD is expected to be reduced or eliminated by genetically modifying the TRAC gene to reduce or eliminate its expression.
  • Engineered cellular receptors such as CARs can be used to redirect T cells to mediate tumor rejection.
  • CARs can be transduced into T cells to generate CAR-T cells using retroviral, lentiviral, or other integrating vectors.
  • retroviral, lentiviral, or other integrating vectors because viral-mediated transduction results in random integration in the genome, application of such CAR-T cells may be limited by risks such as variegated expression, transcriptional silencing, or even oncogenic transformation.
  • Recent advances in genome editing enables target specific engineering in human genomes. For example, programmable nucleases such as CRISPR–Cas9 make double-strand DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions (indels) at specific target sites.
  • DSBs double-strand DNA breaks
  • DSBs are associated with undesired outcomes, including complex mixtures of products and translocations.
  • compositions and methods for targeted delivery of transgenes and precise disruption of the TRAC gene without introducing DSBs are associated with undesired outcomes, including complex mixtures of products and translocations.
  • a prime editing composition or system comprising (A) a first prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first PEgRNA and (B) a second PEgRNA or one or more polynucleotides encoding the second PEgRNA, wherein the first PEgRNA comprises: (i) a first spacer that is complementary to a first search target sequence on a first strand of a TRAC gene, (ii) a first gRNA core capable of binding to a Cas9 protein; and (iii) a first extension arm comprising a first editing template and a first primer binding site (PBS), wherein the first spacer comprises at its 3’ end nucleotides 4-20 of a sequence selected from the group consisting of SEQ ID NOs: 4, 61, 88, and 150 and wherein the first PBS comprises at its 5’ end
  • PBS primer binding site
  • the selected sequence for the first spacer is SEQ ID NO: 4 or 88. [0007] In some embodiments, the selected sequence for the first spacer is SEQ ID NO: 88. [0008] In some embodiments, the selected sequence for the second spacer is SEQ ID NO: 177, 368, or 522. [0009] In some embodiments, the selected sequence for the second spacer is SEQ ID NO: 177. [0010] In some embodiments, the first spacer and/or the second spacer is from 16 to 22 nucleotides in length. [0011] In some embodiments, the first spacer and/or the second spacer is 20 nucleotides in length and comprises the selected sequence.
  • the first PBS is 8-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3- 17, 2-17, or 1-17 of the selected sequence for the first spacer.
  • WSGR Docket No.59761-772601 [0013] In some embodiments, the first PBS is 8-13 nucleotides in length. [0014] In some embodiments, the first PBS is 10, 11, or 12 nucleotides in length.
  • the second PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17, 10-17, 9-17, 8-17, 7-17, 6-17, 5- 17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the second spacer.
  • the second PBS is 8-13 nucleotides in length.
  • the second PBS is 11, 12, or 13 nucleotides in length.
  • the first gRNA core and the second gRNA core comprise the same sequence.
  • the first gRNA core, the second gRNA core, or both comprise SEQ ID NO: 590.
  • the first spacer, the first gRNA core, the first editing template, and the first PBS form a contiguous sequence in a single molecule.
  • the first PEgRNA comprises from 5’ to 3’ the first spacer, the first gRNA core, the first editing template, and the first PBS.
  • the second spacer, the second gRNA core, the second editing template, and the second PBS form a contiguous sequence in a single molecule.
  • the second pegRNA comprises from 5’ to 3’ the second spacer, the second gRNA core, the second editing template, and the second PBS.
  • the first editing template comprises a region of complementarity to the second editing template.
  • the first editing template and the second editing template each encodes all or a fragment of a recombinase recognition sequence (RSS) or the reverse complement thereof, wherein the first editing template encodes at least a 5’ portion of the RSS or the reverse complement thereof, wherein the second editing template encodes at least a 3’ portion of the RSS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second editing templates have perfect reverse complementarity to each other.
  • RSS recombinase recognition sequence
  • the first editing template encodes the RSS.
  • the second editing template encodes the RSS.
  • the RSS is an attB sequence recognized by a Bxb1 recombinase.
  • the RSS is an attP sequence recognized by a Bxb1 recombinase.
  • the RSS is an attB sequence recognized by a Pa01 recombinase.
  • WSGR Docket No.59761-772601 [0032]
  • the RSS is an attP sequence recognized by a Pa01 recombinase.
  • the first editing template comprises an RTT #1 from Table 6 and the second editing template comprises an RTT #2 from the same RTT Pair in Table 6, or wherein the first editing template comprises an RTT #2 from Table 6 and the second editing template comprises an RTT #1 from the same RTT Pair in Table 6.
  • the first editing template comprises SEQ ID NO: 23 and the second editing template comprises SEQ ID NO: 196.
  • the first editing template comprises SEQ ID NO: 24 and the second editing template comprises SEQ ID NO: 106.
  • the first editing template comprises SEQ ID NO: 27 and the second editing template comprises SEQ ID NO: 107.
  • first editing template comprises SEQ ID NO: 106 and the second editing template comprises SEQ ID NO: 24.
  • the first editing template comprises SEQ ID NO: 107 and the second editing template comprises SEQ ID NO: 27.
  • the first editing template comprises SEQ ID NO: 25 and the second editing template comprises SEQ ID NO: 197.
  • the first editing template comprises SEQ ID NO: 28 and the second editing template comprises SEQ ID NO: 199.
  • the first editing template comprises SEQ ID NO: 22 and the second editing template comprises SEQ ID NO: 195.
  • the first editing template comprises SEQ ID NO: 26 and the second editing template comprises SEQ ID NO: 198.
  • the first editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the second editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the first editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second editing templates have perfect reverse complementarity to each other.
  • at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second editing templates have prefect reverse complementarity to each other.
  • the length of the region of complementarity of the first editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the first editing template, optionally wherein the length of the region of complementarity of the first editing template is at least 52%, at least 53%, or at least 55% of the length of the first editing template.
  • the length of the region of complementarity of the second editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the second editing template, optionally wherein the length of the region of complementarity of the second editing template is at least 52%, at least 53%, or at least 55% of the length of the second editing template.
  • the first spacer comprises SEQ ID NO: 4, and the first PBS comprises SEQ ID NO: 13, or the first spacer comprises SEQ ID NO: 88, and the first PBS comprises SEQ ID NO: 96, or the first spacer comprises SEQ ID NO: 88, and the first PBS comprises SEQ ID NO: 157, or the first spacer comprises SEQ ID NO: 88, and the first PBS comprises SEQ ID NO: 99, or the first spacer comprises SEQ ID NO: 88, and the first PBS comprises SEQ ID NO: 97; and (b) the second spacer comprises SEQ ID NO: 177, and the second PBS comprises SEQ ID NO: 188, or the second spacer comprises SEQ ID NO: 368, and the second PBS comprises SEQ ID NO: 376.
  • the first spacer SEQ ID NO: 4 has the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15; or the first spacer comprises SEQ ID NO: 88, and the first PBS has the sequence according to SEQ ID NO: 96 or SEQ ID NO: 98; and (b) the second spacer comprises SEQ ID NO: 177, and the second PBS has the sequence according to SEQ ID NO: 186 or SEQ ID NO: 188, the second spacer comprises SEQ ID NO: 368, and the second PBS has the sequence according to SEQ ID NO: 373 or SEQ ID NO: 374; or the second spacer comprises SEQ ID NO: 522, and the second PBS has the sequence according to SEQ ID NO: 531 or 533.
  • the first spacer comprises SEQ ID NO: 88, and the first PBS has the sequence according to SEQ ID NO: 96, and wherein the second spacer comprises SEQ ID NO: 177, and the second PBS has the sequence according to SEQ ID NO: 186.
  • the first editing template comprises SEQ ID NO: 27 and the second editing template comprises SEQ ID NO: 107.
  • the first editing template comprises SEQ ID NO: 107 and the second editing template comprises SEQ ID NO: 27.
  • the first PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 36, 118, 123, and 1132-1134; and wherein the second PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1135-1142.
  • WSGR Docket No.59761-772601 In some embodiments, the first PEgRNA comprises SEQ ID NO: 118 and the second PEgRNA comprises SEQ ID NO: 1140.
  • the first PEgRNA comprises SEQ ID NO: 136 and the second PEgRNA comprises SEQ ID NO: 224.
  • the first PEgRNA comprises SEQ ID NO: 51 and the second PEgRNA In some embodiments, the first PEgRNA comprises SEQ ID NO: 126 and the second PEgRNA comprises SEQ ID NO: 220. [0057] In some embodiments, the first PEgRNA comprises SEQ ID NO: 44 and the second PEgRNA comprises SEQ ID NO: 550. [0058] In some embodiments, the first PEgRNA comprises SEQ ID NO: 111 and the second PEgRNA comprises SEQ ID NO: 1251.
  • the first PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 36, 38, 39, 43, 44, 49, 50, 51, 52, 53, 79, 80, 82, 109, 111, 112, 114, 115, 118, 120, 122, 123, 125, 126, 129, 130, 134, 135, 136, 140, 141, 143, 168, 169, 171, 592, 593, 594, and 1132; and wherein the second PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 203, 207, 210, 211, 212, 215, 217, 219, 220, 221, 222, 224, 226, 228, 251, 252, 254, 278, 279, 281, 305, 306, 308, 332, 333, 335, 359, 360, 362, 388, 390, 392, 395, 398, 397,
  • the first PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 30, 44, 109, and 126; and wherein the second PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 207, 221, 388, and 400.
  • the first PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 136, 141, 51, and 53; and wherein the second PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 226, 403, 558, 556, and 224.
  • the first PEgRNA comprises SEQ ID NO: 136
  • the second PEgRNA comprises SEQ ID NO: 224.
  • the first PEgRNA and/or the second PEgRNA further comprises a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the first PBS or the second PBS via a linker.
  • the first PEgRNA and/or the second PEgRNA further comprises 5’mN*mN*mN* and 3’ mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • the prime editing composition or system of any one of the embodiments herein further comprises a prime editor or one or more polynucleotides encoding the WSGR Docket No.59761-772601 prime editor, wherein the prime editor comprises (a) a Cas9 nickase having a nuclease inactivating mutation in a HNH domain and (b) a reverse transcriptase.
  • the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1007.
  • the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1003.
  • the prime editor is a fusion protein.
  • the fusion protein comprises SEQ ID NO: 1033.
  • the one or more polynucleotides encoding the prime editor comprise (a) a first sequence encoding an N-terminal portion of the Cas9 nickase and an intein-N and (b) a second sequence encoding an intein-C, a C-terminal portion of the Cas9 nickase, and the reverse transcriptase.
  • the prime editing composition or system of any one of the embodiments herein further comprises a recombinase that recognizes the one or more recombinase recognition sequences (RSSs) or one or more polynucleotides encoding the recombinase.
  • the recombinase is Bxb1 or Pa01.
  • the recombinase is a Bxb1 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1131.
  • the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment.
  • the recombinase is fused or linked to the prime editor.
  • the prime editing composition or system of any one of the embodiments herein further comprises a DNA polynucleotide that comprises (a) a donor sequence and (b) a second RSS recognized by the recombinase.
  • the RSS comprises a Bxb1 attB sequence provided in Table 5, and the second RSS comprises a corresponding attP sequence provided in Table 5, or
  • the RSS comprises a Bxb1 attP sequence provided in Table 5, and the second RSS comprises a corresponding attB sequence provided in Table 5.
  • the RSS sequence comprises SEQ ID NO: 1187
  • the second RSS comprises SEQ ID NO: 1188.
  • the donor sequence comprises an open reading frame that encodes a polypeptide. [0080] In some embodiments, the donor sequence encodes a chimeric antigen receptor (CAR). [0081] In some embodiments, the donor sequence encodes a CD19 CAR. [0082] In some embodiments, the donor sequence comprises a splice acceptor sequence.
  • the prime editing composition or system of any one of the embodiments herein comprises one or more vectors that comprises the one or more polynucleotides encoding the first PEgRNA, the one or more polynucleotides encoding the second PEgRNA, and the one or more polynucleotides encoding the prime editor.
  • the prime editing composition or system of any one of the embodiments herein comprises one or more vectors that comprises the one or more polynucleotides encoding the first PEgRNA, the one or more polynucleotides encoding the second PEgRNA, the one or more polynucleotides encoding the prime editor, the one or more polynucleotides encoding the recombinase, and the donor sequence.
  • the one or more vectors are AAV vectors.
  • the one or more polynucleotides encoding the prime editor and/or the one or more polynucleotides encoding the recombinase are mRNA.
  • the prime editing composition or system of any one of the embodiments herein further comprises a third PEgRNA or a nucleic acid encoding the third PEgRNA, wherein the third PEgRNA comprises: (i) a third spacer that is complementary to a search target sequence on a first strand of a ⁇ 2-microglobulin (B2M) gene; (ii) a third gRNA core capable of binding to a Cas9 protein; and (iii) a third extension arm comprising: (a) a third editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and (b) a third primer binding site (PBS) that comprises a sequence that is a reverse complement of a portion of the
  • the third spacer comprises at its 3’ end SEQ ID NO: 1063.
  • the third PBS comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1063.
  • the editing template encodes one or more in-frame stop codons or the complement thereof in the B2M gene.
  • the one or more in-frame stop codons comprise a nonsense mutation in the B2M gene.
  • the one or more in-frame stop codons comprise an insertion in the B2M gene.
  • the editing template encodes an insertion of two in-frame stop codons in the B2M gene.
  • the insertion is TAATAA or TTATTA.
  • the editing template encodes a frameshift mutation in the B2M gene.
  • the frameshift mutation is an insertion.
  • WSGR Docket No.59761-772601 In some embodiments, the insertion is c.50insG or the complement thereof.
  • the frameshift mutation is a deletion. [0099] In some embodiments, the deletion is c.51delC or the complement thereof.
  • the prime editing composition or system further comprises a third PEgRNA or a nucleic acid encoding the third PEgRNA, wherein the third PEgRNA comprises: a. a third spacer comprising at its 3’ end SEQ ID NO: 1063; b. a third gRNA core capable of binding to a Cas9 protein; and c. a third extension arm comprising: i. a third editing template comprising at its 3’ end: (A) nucleotides 13-24 of SEQ ID NO: 1079, (B) nucleotides 12-20 of SEQ ID NO: 1085, or (C) nucleotides 7-17 of SEQ ID NO: 1089, and ii.
  • the third PEgRNA comprises: a. a third spacer comprising at its 3’ end SEQ ID NO: 1063; b. a third gRNA core capable of binding to a Cas9 protein; and c. a third extension arm comprising: i. a third editing template comprising at its 3’ end
  • a third primer binding site comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1063.
  • the third spacer is from 17-22 nucleotides in length.
  • the third spacer comprises at its 3’ end any one of SEQ ID NOs: 1060- 1063.
  • the third editing template comprises at its 3’ end nucleotides 13-24 of SEQ ID NO: 1077.
  • the third editing template comprises at its 3’ end SEQ ID NO: 1077.
  • the third editing template comprises at its 3’ end any one of SEQ ID NOs: 1078 or 1079. [0106] In some embodiments, the third editing template comprises at its 3’ end nucleotides 12-20 of SEQ ID NO: 1085. [0107] In some embodiments, the third editing template comprises at its 3’ end SEQ ID NO: 1081. [0108] In some embodiments, the third editing template comprises at its 3’ end any one of SEQ ID Nos: 1080, 1082-1085. [0109] In some embodiments, the third editing template comprises at its 3’ end nucleotides 7-17 of SEQ ID NO: 1089.
  • the third editing template comprises at its 3’ end any one of SEQ ID NOs: 1087-1089. [0111] In some embodiments, the third editing template has a length of 24 nucleotides or less. [0112] In some embodiments, the third editing template has a length of 20 nucleotides or less. [0113] In some embodiments, the third editing template has a length of 10 to 20 nucleotides. [0114] In some embodiments, the third editing template has a length of 12 to 20 nucleotides. [0115] In some embodiments, third editing template has a length of 11 to 17 nucleotides. [0116] In some embodiments, the third editing template is 16 to 24 nucleotides in length.
  • the third PBS comprises at its 3’ end a sequence set forth in any one of sequence numbers 1064-1076. [0118] In some embodiments, the third PBS has a length of 17 nucleotides or less. WSGR Docket No.59761-772601 [0119] In some embodiments, the third PBS is 8 to 15 nucleotides in length. [0120] In some embodiments, the third PBS is 8 to 14 nucleotides in length. [0121] In some embodiments, the third PBS is 12 nucleotides in length.
  • the third spacer, the third gRNA core, the third editing template, and the third PBS form a contiguous sequence in a single molecule.
  • the single molecule comprises from 5’ to 3’, the third spacer, the third gRNA core, the third editing template and the third PBS.
  • the third PEgRNA comprises a sequence selected from any one SEQ ID NOs: 1090-1120.
  • the prime editing composition or system of any one of the embodiments herein further comprises a nick guide RNA (ngRNA), or a nucleic acid encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer that is complementary to a ngRNA target sequence on the second strand of the B2M gene; and (ii) an ngRNA core capable of binding a Cas9 protein.
  • the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1121-1126.
  • ngRNA spacer comprises at its 3’ end any one of SEQ ID NOs: 1121- 1126. [0128] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NO: 1126. [0129] In some embodiments, the one or more nucleotide encoded by the editing template is c.51delC or the complement thereof, and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1124.
  • the one or more nucleotide encoded by the editing template is c.50insG or the complement thereof, and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1125 or 1126.
  • the ngRNA comprises SEQ ID NO: 1129.
  • an LNP comprising the prime editing composition or system of any one of the embodiments herein.
  • a pharmaceutical composition comprising the prime editing composition or system of any one of the embodiments herein or the LNP of any one of the embodiments herein and a pharmaceutically acceptable excipient.
  • a method of editing a TRAC gene comprising contacting the TRAC gene with (a) the prime editing composition or system of any one of the embodiments herein and a prime editor comprising a Cas9 nickase having a nuclease inactivation mutation in a HNH domain and a reverse transcriptase or (b) the prime editing composition or system of any one of the embodiments herein.
  • the method further comprises contacting the TRAC gene with a recombinase or one or more polynucleotides encoding the recombinase and a DNA polynucleotide comprising (a) a donor sequence and (b) one or more recombinase recognition sequences recognized by the recombinase.
  • a method of inserting a donor sequence into a TRAC gene the method comprises contacting the TRAC gene with the prime editing composition or system of any one of the embodiments herein or the LNP of any one of the embodiments herein.
  • a method of making a modified cell comprising contacting a cell with the prime editing composition or system of any one of the embodiments herein or the LNP of any one of the embodiments herein.
  • the TRAC gene is in a cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is an immune cell, optionally wherein, the cell is a T cell.
  • the cell is in a subject.
  • the cell is from a subject.
  • the subject is a human.
  • the method of any one of the embodiments herein further comprises editing a B2M gene.
  • the editing comprises contacting the B2M gene with the prime editing composition or system of any one of the embodiments herein and a prime editor comprising a Cas9 nickase having a nuclease inactivation mutation in a HNH domain and a reverse transcriptase.
  • an edited TRAC gene that comprises GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1046) and/or GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1047) relative to a wildtype TRAC gene.
  • the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1046), a donor sequence, and GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1047).
  • the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1047), a donor sequence, and GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1046).
  • the donor sequence encodes a chimeric antigen receptor (CAR), optionally wherein the donor encodes a CD19 CAR.
  • the insert sequence is between a first chromosome location and a second chromosome location, wherein the first chromosome location is selected from the group consisting of human chromosome 14 positions 22547458, 22547457, 22547449, and 22547448, and wherein the second chromosome location is selected from the group consisting of human chromosome 14 positions 22547533, 22547523, 22547491, 22547528, 22547497, 22547579, 22547522, 22547485, 22547506, 22547560, 22547505, 22547529, and 22547490.
  • the insert sequence is between human chromosome 14 positions 22547458 and 22547533. [0154] In some embodiments, the insert sequence is between human chromosome 14 positions 22547458 and 22547522. [0155] In some embodiments, the insert sequence is between human chromosome 14 positions 22547458 and 22547529. [0156] In some embodiments, the insert sequence is between human chromosome 14 positions 22547449 and 22547533. [0157] In some embodiments, the insert sequence is between human chromosome 14 positions 22547449 and 22547522. [0158] In some embodiments, the insert sequence is between human chromosome 14 positions 22547449 and 22547529.
  • the T cell further comprises premature stop codon relative to a wildtype B2M gene.
  • the T cell further comprises a B2M gene comprising a c.51delC edit relative to a wildtype B2M gene.
  • the T cell further comprises a B2M gene comprising a c.50insG edit relative to a wildtype B2M gene.
  • the T cell further comprises a B2M gene comprising a c.54insTAATAA edit relative to a wildtype B2M gene.
  • the human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38).
  • a prime editing composition or system comprising (a) a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: (i) a spacer that is complementary to a search target sequence on a first strand of a target gene, (ii) a gRNA core capable of binding to a Cas9 nickase, and (iii) an extension arm comprising a primer binding site (PBS) that comprises a region of complementarity to a second strand of the target gene and an editing template encoding a recombinase recognition sequence (RSS) WSGR Docket No.59761-772601 recognized by a Pa01 recombinase; (b) a prime editor comprising the
  • the PBS comprises a region of complementarity to a region upstream of the nick site.
  • the editing template comprises a region of complementarity to a region downstream of the nick site.
  • the Cas9 nickase comprises a nuclease inactivation mutation in a HNH domain.
  • a prime editing composition or system comprising (A) a first prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first PEgRNA, (B) a second PEgRNA or one or more polynucleotides encoding the second PEgRNA, (C) a prime editor comprising a Cas9 nickase having a nuclease inactivation mutation in a HNH domain and a reverse transcriptase, or one or more polynucleotides encoding the prime editor, and (D) a Pa01 recombinase or one or more polynucleotides encoding the Pa01 recombinase, wherein the first PEgRNA comprises: (i) a first spacer that is complementary to a first search target sequence on a first strand of a target gene, (ii) a first gRNA core capable of binding to the Cas9 nickase; and
  • the first editing template encodes the RSS.
  • the second editing template encodes the RSS.
  • the first editing template comprises a 5’ fragment of an RTT listed in RTT pair 2 or 3 of Table 6 and wherein the second editing template comprises a full length or 5’ WSGR Docket No.59761-772601 fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of an RTT listed in RTT pair 2 or 3 of Table 6 and wherein the first editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the first PBS comprises a region of complementarity to a region upstream of a nick site in the second strand
  • the second PBS comprises a region of complementarity to a region upstream of a nick site in the first strand.
  • the prime editing composition or system of any one of the embodiments herein further comprises a DNA polynucleotide that comprises (i) a second RSS recognized by the Pa01 recombinase, and (ii) a donor sequence.
  • the target gene is TRAC.
  • a method for integrating a donor sequence in a target gene comprising contacting the target gene with the prime editing composition or system of any one of the embodiments herein.
  • FIG.1 depicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double- stranded target DNA sequence.
  • PEgRNA prime editing guide RNA
  • FIG.2 depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor.
  • FIG.3 is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown.
  • FIG.4A depicts an exemplary schematic of a dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between sequences.
  • FIG.4B depicts an exemplary schematic of dual prime editing with a replacement duplex (RD) comprising an overlap duplex (OD).
  • RD replacement duplex
  • OD overlap duplex
  • FIG.4C depicts an exemplary schematic of dual prime editing. Same color/shading indicates complementarity or identity between sequences.
  • FIG.4D depicts an exemplary schematic of dual prime editing. Same color/shading indicates complementarity or identity between sequences.
  • FIG.4E depicts an exemplary schematic of dual prime editing. Same color/shading indicates complementarity or identity between sequences.
  • FIG.4F depicts an exemplary schematic of dual prime editing. Same color/shading indicates complementarity or identity between sequences.
  • FIG.4G depicts an exemplary schematic of dual prime editing. Same color/shading indicates complementarity or identity between sequences.
  • compositions provided herein can comprise prime editors (PEs) that may use engineered guide polynucleotides, e.g., prime editing guide RNAs (PEgRNAs), that can direct PEs to specific DNA targets and can encode DNA edits on the target gene TRAC that serve a variety of functions, including disruption of the target gene (e.g., by introducing one or mutation in the target gene), introduction of exogenous sequences in to the TRAC gene (e.g., one or more recombinase recognition sequences), and integration of DNA donors in the gene (e.g., expression cassettes, ORFs).
  • PEs prime editors
  • PEgRNAs prime editing guide RNAs
  • compositions that comprise edited cells generated by the methods disclosed herein.
  • the following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.
  • a “cell” generally refers to a biological cell.
  • a cell can be the basic structural, functional and/or biological unit of a living organism.
  • a cell can originate from any organism having one or more cells. Sometimes a cell may not originate from a natural organism (e.g., a cell can be synthetically made, sometimes termed an artificial cell).
  • the cell is a human cell.
  • a cell may be of or derived from different tissues, organs, and/or cell types.
  • the cell is a primary cell.
  • primary cell means a cell isolated from an organism, e.g., a mammal, which is grown in tissue culture (i.e., in vitro) for the first time before subdivision and transfer to a subculture.
  • tissue culture i.e., in vitro
  • mammalian cells, including primary cells and stem cells can be modified through introduction of one or more polynucleotides, polypeptides, and/or prime editing compositions (e.g., through transfection, transduction, electroporation and the like) and further passaged.
  • Such modified cells include a T-cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de-differentiated cell thereof, and stem cells.
  • a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer
  • a cell is a naive T cell (e.g., a naive CD8+ T cell). In some embodiments, the cell is a transformed T cell. In some embodiments, the cell is an immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the cell is a T-cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a human T cell, or a progenitor or a precursor WSGR Docket No.59761-772601 thereof.
  • the cell is a T helper cell (e.g., Th1 cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, and Tfh (follicular helper) cell).
  • the cell is a cytotoxic T cell.
  • the cell is a CD8+ T cell.
  • the cell is a CD4+ T cell.
  • the cell is a memory T cell (e.g., (e.g., central memory T cell (T CM ), stem memory T cell (TSCM), effector memory T cell, Tissue resident memory T cell).
  • the cell is an effector memory T cell (e.g., T EM cells and T EMR A (CD45RA + ) cells).
  • the cell is a regulatory T cell.
  • the cell is a natural killer T cell.
  • the cell is a Mucosal associated invariant T cell.
  • the cell is a ⁇ T cell.
  • the cell is an effector T cell.
  • the cell is a thymocyte.
  • the cell is a lymphoid cell.
  • the cell is a common lymphoid progenitor cells.
  • the cell is an early thymic progenitor cell.
  • the cell is a CD3+ cell. In some embodiments, the cell is a tumor infiltrating lymphocyte. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is a plasma cell. In some embodiments, the cell is an activated T cell. [0201] In some embodiments, the cell is a stem cell (e.g., adult stem cell, embryonic stem cell, non- embryonic stem cell), cord blood stem cell, progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell).
  • stem cell e.g., adult stem cell, embryonic stem cell, non- embryonic stem cell
  • cord blood stem cell e.g., progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell.
  • the cell is a pluripotent cell (e.g., a pluripotent stem cell).
  • the cell e.g., a stem cell
  • the cell is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell.
  • the cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the cell is a hematopoietic stem cell.
  • the cell is a hematopoietic stem and progenitor cell.
  • the cell is a multipotent progenitor cell.
  • the cell is a T-cell progenitor.
  • the cell is a T-cell precursor. In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human pluripotent stem cell. In some embodiments, the cell is a non-embryonic stem cell. In some embodiments, the cell is an induced human pluripotent stem cell. In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human T- cell progenitor. In some embodiments, the cell is a human T-cell precursor. [0202] In some embodiments, the cell is a mammalian cell.
  • a cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal.
  • the cell is a differentiated cell.
  • the cell is differentiated from an induced pluripotent stem cell.
  • the cell is a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated WSGR Docket No.59761-772601 invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell differentiated from an iPSC, ESC, a T-cell precursor, or a T-cell progenitor.
  • a primary T cell e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated WSGR Docke
  • the cell is a differentiated human cell.
  • the cell is differentiated from an induced human pluripotent stem cell.
  • the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T- cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell.
  • a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T- cell, a CD8+ T cell, a memory T cell
  • the cell is in a subject, e.g., a human subject.
  • the cell is obtained from a subject prior to editing.
  • the cell is obtained from a patient having a cancer, a microbial infection, a graft vs host disease, or an autoimmune disorder.
  • the cell Prior to editing by the methods and compositions disclosed herein the cell can be obtained from a subject through a variety of non-limiting methods.
  • T cells can be obtained from a number of non- limiting sources, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • the cell can be obtained from a cell bank, a blood bank, cell culture, or any number of T cell lines available, and known to those skilled in the art.
  • Cells may also be obtained from a tissue biopsy, surgery, blood, plasma, serum, or other biological fluid.
  • the cell can be obtained prior to editing from one or more healthy donors, from a patient having a cancer, a microbial infection, a graft versus host infection, or an autoimmune disorder.
  • a cell can be obtained (i.e., isolated or purified) prior to editing from a whole blood sample by lysing red blood cells or a fractionated blood sample, and removing peripheral mononuclear blood cells by centrifugation.
  • the cell can be further isolated or purified using a selective purification method that isolates the cell based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO (e.g., by flow cytometry).
  • CD4+ is used as a marker to select T cells.
  • CD8+ is used as a marker to select T cells.
  • CD4+ and CD8+ are used as a marker to select regulatory T cells.
  • the edited cell produced using the methods and compositions disclosed herein are cultured, proliferated, expanded, differentiated, and or de-differentiated in vitro.
  • the cell comprises a prime editor or a prime editing composition.
  • the cell comprises a dual prime editing composition or system comprising a prime editor and at least two PEgRNAs that are different from each other.
  • the cell is from a human subject.
  • the cell is from a human subject, and comprises a prime editor or a prime editing composition for editing a TRAC gene.
  • the cell WSGR Docket No.59761-772601 is from a human subject and the TRAC gene has been edited by prime editing.
  • the cell comprises a prime-edited TRAC gene, and is administered to a subject (e.g., a human subject).
  • the cell is in a human subject, and comprises a prime editor or a prime editing composition for editing a TRAC gene.
  • the cell is from the human subject and the TRAC gene has been edited or corrected by prime editing.
  • the human subject is a healthy donor, or has a disease, disorder, or a condition, e.g., a cancer, a microbial infection, an autoimmune disorder, a T cell malignancy, or a graft versus host disorder.
  • the human subject is in need of, or is undergoing, or will be undergoing an immune cell immunotherapy (e.g., a T cell therapy such as a CAR-T cell therapy).
  • an immune cell immunotherapy e.g., a T cell therapy such as a CAR-T cell therapy.
  • the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.
  • protein and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three- dimensional conformation.
  • a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody. In some embodiments, a protein may be a full-length protein (e.g., a fully processed protein having certain biological function).
  • a protein may be a variant or a fragment of a full-length protein.
  • a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring S. pyogenes Cas9 protein.
  • a variant of a protein or enzyme for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
  • a protein comprises one or more protein domains or subdomains.
  • polypeptide domain when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
  • a protein comprises multiple protein domains.
  • a protein comprises multiple protein domains that are naturally occurring.
  • a protein WSGR Docket No.59761-772601 comprises multiple protein domains from different naturally occurring proteins.
  • a prime editor may be a fusion protein comprising a Cas9 protein domain of S.
  • a protein comprises a functional variant or functional fragment of a full-length wild-type protein.
  • a “functional fragment” or “functional portion”, as used herein, refers to any portion of a reference protein (e.g., a wild-type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • a functional fragment of a reverse transcriptase may encompass less than the entire amino acid sequence of a wild-type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide.
  • a functional fragment thereof may retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas9 may encompass less than the entire amino acid sequence of a wild-type Cas9, but retains its DNA binding ability and lacks its nuclease activity partially or completely.
  • a “functional variant” or “functional mutant”, as used herein, refers to any variant or mutant of a reference protein (e.g., a wild-type protein) that encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions.
  • a functional variant of a reverse transcriptase may comprise one or more amino acid substitutions compared to the amino acid sequence of a wild-type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide.
  • a functional variant thereof may retain one or more of the functions of at least one of the functional domains.
  • a functional variant of a Cas9 may comprise one or more amino acid substitutions in a nuclease domain, e.g., an H840A amino acid substitution, compared to the amino acid sequence of a wild-type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely.
  • the term “function” and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose.
  • functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose.
  • the term functional WSGR Docket No.59761-772601 may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.
  • a protein or polypeptide includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
  • a protein or polypeptides includes non-naturally occurring amino acids (e.g., amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics).
  • a protein or polypeptide is modified.
  • a protein comprises an isolated polypeptide.
  • isolated means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.
  • a protein is present within a cell, a tissue, an organ, or a virus particle.
  • a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell).
  • the cell is in a tissue, in a subject, or in a cell culture.
  • the cell is a microorganism (e.g., a bacterium, fungus, protozoan, or virus).
  • a protein is present in a mixture of analytes (e.g., a lysate).
  • the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.
  • the terms “homologous,” “homology,” or “percent homology” as used herein refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence or a polynucleotide sequence and a corresponding reference polynucleotide sequence.
  • Homology can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, a “homologous sequence” of nucleic acid sequences may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence.
  • a "region of homology to a genomic region” can be a region of DNA that has a similar sequence to a given genomic region in the genome.
  • a region of homology can be of any length that is sufficient to promote binding of a spacer, or a primer binding site to the complementary sequence of a genomic region.
  • the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases WSGR Docket No.59761-772601 in length such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.
  • sequence homology or identity when a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length.
  • Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al, J. Mol. Biol.215:403- 410, 1990.
  • BLAST Basic Local Alignment Search Tool
  • a publicly available, internet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences”, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins” J. Mol.
  • Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https://fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length.
  • polynucleotide or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA.
  • a polynucleotide is double-stranded, e.g., a double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • Polynucleotides can have any three-dimensional structure.
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).
  • a polynucleotide may be modified.
  • the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
  • modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
  • the modification may be on the internucleoside linkage (e.g., phosphate backbone).
  • multiple WSGR Docket No.59761-772601 modifications are included in the modified nucleic acid molecule.
  • a single modification is included in the modified nucleic acid molecule.
  • complement refers to the ability of two polynucleotide molecules to base pair with each other.
  • Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • hydrogen bonding may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • an adenine on one polynucleotide molecule will base pair to a thymine or an uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to guanine on a second polynucleotide molecule.
  • Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence.
  • the two DNA molecules 5’-ATGC-3’ and 5'-GCAT-3’ are complementary, and the complement of the DNA molecule 5’-ATGC-3’ is 5’-GCAT-3’.
  • a percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule.
  • substantially complementary refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides.
  • “Substantial complementary” can also refer to a 100% complementarity over a portion or a region of two polynucleotide molecules.
  • the portion or the region of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which polynucleotides, e.g., the transcribed mRNA, are translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of the protein encoded by the gene after transcription and translation of the gene.
  • expression of a polynucleotide is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a gene is determined by the amount of the mRNA, or transcript that is encoded by the gene after transcription of the gene. In some embodiments, expression of a polynucleotide, e.g., an mRNA, is determined by the amount of the protein encoded by the mRNA after translation of the mRNA.
  • expression of a polynucleotide is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide.
  • sampling may comprise capillary sequencing, bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high- throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof.
  • encode refers to a polynucleotide which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as a polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof.
  • a polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid.
  • a polynucleotide comprises one or more codons that encode a polypeptide.
  • a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide.
  • the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.
  • mutation refers to a change and/or alteration in an amino acid sequence of a protein or nucleic acid sequence of a polynucleotide. Such changes and/or alterations may comprise the substitution, insertion, deletion and/or truncation of one or more amino acids, in the case of an amino acid sequence, and/or nucleotides, in the case of nucleic acid sequence, compared to a reference amino acid or a reference nucleic acid sequence.
  • the reference sequence is a wild-type sequence.
  • a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide.
  • the mutation in the amino acid sequence of the polypeptide or the mutation in the nucleic acid sequence of the polynucleotide is a mutation associated with a disease state.
  • the term “subject” and its grammatical equivalents as used herein may refer to a human or a non-human.
  • a subject may be a mammal.
  • a human subject may be male or female.
  • a human subject may be of any age.
  • a subject may be a human embryo.
  • a human subject may be a newborn, an infant, a child, an adolescent, or an adult.
  • a human subject may be in need of treatment for a genetic disease WSGR Docket No.59761-772601 or disorder.
  • a human subject may be in need of a cell therapy., e.g., an immune cell immunotherapy such as a T cell therapy).
  • a human subject may be in need of a CAR-T cell therapy.
  • treatment or “treating” and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder.
  • Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
  • Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
  • this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
  • Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
  • a condition may be pathological.
  • a treatment may not completely cure or prevent a disease, condition, or disorder.
  • a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder.
  • a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.
  • the term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • the terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time.
  • Prevent also means reducing risk of developing a disease, disorder, or condition.
  • Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
  • a composition e.g., a pharmaceutical composition, prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.
  • an effective amount refers to a quantity of a composition, for example a prime editing composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein.
  • An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is in vitro, ex vivo or in vivo.
  • An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation observed relative to a negative control.
  • An effective amount or dose can induce, for example, about 2-fold increase, about 3-fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, WSGR Docket No.59761-772601 about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25- fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500-fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation.
  • the amount of target gene modulation may be measured by any suitable method known in the art.
  • the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient.
  • an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest (e.g., a TRAC gene) in a cell (e.g., a cell in vitro ⁇ ex vivo or in vivo).
  • a “therapeutically effective amount” refers to a quantity of a composition comprising the edited cells that can be sufficient to result in a desired activity upon introduction into a subject (e.g., a human subject).
  • construct refers to a polynucleotide or a portion of a polynucleotide, comprising one or more nucleic acid sequences encoding one or more transcriptional products and/or proteins.
  • a construct may be a recombinant nucleic acid molecule or a part thereof.
  • the one or more nucleic acid sequences of a construct are operably linked to one or more regulatory sequences, for example, transcriptional initiation regulatory sequences.
  • a construct is a vector, a plasmid, or a portion thereof.
  • a construct a construct comprises DNA.
  • a construct comprises RNA.
  • a construct is double-stranded. In some embodiments, a construct is single-stranded. In some embodiments, a construct comprises an expression cassette.
  • An expression cassette means a polynucleotide comprising a nucleic acid sequence that encodes one or more transcriptional products and is operably linked to at least one transcriptional regulatory sequence, e.g., a promoter.
  • exogenous when used in reference to a biomolecule, e.g., a polynucleotide sequence or a polypeptide sequence refers to a biomolecule that is not native to a specific biological context, e.g., a gene, a particular chromosome, a particular cell or chromosomal site of the cell, tissue, or organism, or, if from the same source, is modified from its original form or is present in a non- native location, e.g., a chromosome location.
  • endogenous when used in reference to a biomolecule, e.g., a polynucleotide sequence or a polypeptide sequence refers to a biomolecule that is native to or naturally occurring in a specific biological context, e.g., a gene, a particular chromosome, a particular cell or chromosomal site of the cell, tissue, or organism.
  • an endogenous sequence may be a wild-type sequence or may comprise one or more mutations compared to a wild-type sequence.
  • an endogenous sequence is mutated compared to a wild-type sequence and may cause WSGR Docket No.59761-772601 or be associated with a disease or disorder in a subject.
  • a wild- type sequence is a gene sequence found in healthy individuals, wherein the wild-type sequence does not include a mutation causative of the specific disease.
  • the term “recombinase,” as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences.
  • Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases).
  • serine recombinases include, without limitation, Si74, No67, Kp03, Pa01, Nm60, BceINTa, BcytINTd, SscINTd, SacINTd, Hin, Gin, Tn3, I3-six, CinH, ParA, y6, Bxbl, OC31, TP901, TG1, pBT1, R4, pRV1, pFC1, MR11, A118, U153, and gp29.
  • tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2.
  • Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications, as described in international publication no. WO2020191248A1, which is hereby incorporated by reference in its entirety.
  • the recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention.
  • the methods and compositions of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities.
  • the catalytic domains of a recombinase are fused to a programmable DNA binding domain of a prime editor, such as a RNA-programmable nuclease (e.g., dCas9, Cas9 nickase, or a fragment thereof), such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid (e.g., the recombinase domain is engineered such that it does not have specific DNA binding activity).
  • a prime editor such as a RNA-programmable nuclease (e.g., dCas9, Cas9 nickase, or a fragment thereof)
  • serine recombinases of the resolvase-invertase group e.g., Tn3 and 76 resolvases and the Hin and Gin invertases
  • the catalytic domains of these recombinases are thus amenable to being combined with, e.g., fused to or connected to a prime editor or a component thereof, as described herein, e.g., following the isolation of ⁇ activated' recombinase mutants which do not require any accessory factors (e.g., DNA binding activities).
  • recombinase recognition sequence or equivalently as “RRS” or “recombinase target sequence” or “recombinase site,” as used herein, refers to a nucleotide sequence target recognized by a recombinase and which undergoes strand exchange with another DNA molecule having a the RRS that results in excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences.
  • a prime editing composition may install one or more recombinase sites in a target sequence, or in more than one target sequence.
  • the recombinase sites can be installed at adjacent target sites or non-adjacent target sites (e.g., separate chromosomes).
  • single installed recombinase sites can be used as "landing sites" for a recombinase-mediated reaction between the genomic recombinase site and a second recombinase site within an exogenously supplied nucleic acid molecule, e.g., a plasmid or a DNA vector. This enables the targeted integration of a desired nucleic acid molecule.
  • nucleic acid modification e.g., a genomic modification
  • a recombinase protein e.g., an inventive recombinase fusion protein provided herein. Recombination can result in, inter alia, the insertion, inversion, excision, or translocation of nucleic acids, e.g., in or between one or more nucleic acid molecules.
  • Prime Editing and Dual Prime Editing refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target DNA through target-primed DNA synthesis.
  • a target DNA may comprise a double-stranded DNA molecule having two complementary strands.
  • the two complementary strands of a double- stranded target DNA may comprise a first strand that may be referred to as a “target strand” or a “non- edit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.”
  • a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”.
  • the spacer sequence anneals with the target strand at the search target sequence.
  • the target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).”
  • the non-target strand may also be referred to as the “PAM strand”.
  • the PAM strand comprises a protospacer sequence and optionally a protospacer adjacent motif (PAM) sequence.
  • PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene.
  • a PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence.
  • a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise uracil (U) and the protospacer sequence may comprise thymine (T).
  • the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand).
  • a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA.
  • the position of a nick site is a specific position relative to the position of a specific PAM sequence.
  • the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence.
  • the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active RuvC domain and a nuclease inactive HNH domain.
  • the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N. lari Cas9 nickase.
  • the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain.
  • the nick site is 2 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain.
  • a “primer binding site” is a single- stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e. the non-target strand or the edit strand).
  • the PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site.
  • the PEgRNA complexes with and directs a prime editor to bind the search target sequence on the target strand of the double stranded target DNA, and generates a nick at the nick site on the non-target strand of the double stranded target DNA.
  • the PBS is complementary to or substantially complementary to, and can anneal to, a free 3 ⁇ end on the non-target strand of the double stranded target DNA at the nick site.
  • WSGR Docket No.59761-772601 In some embodiments, the PBS annealed to the free 3 ⁇ end on the non-target strand can initiate target- primed DNA synthesis.
  • An “editing template” of a PEgRNA is a single-stranded portion of the PEgRNA that is 5 ⁇ of the PBS and which encodes a single strand of DNA.
  • the editing template may comprise a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target DNA.
  • the editing template and the PBS are immediately adjacent to each other.
  • a PEgRNA in prime editing comprises a single-stranded portion that comprises the PBS and the editing template immediately adjacent to each other.
  • the single stranded portion of the PEgRNA comprising both the PBS and the editing template is complementary or substantially complementary to an endogenous sequence on the PAM strand (i.e., the non-target strand or the edit strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit position(s).
  • the relative positions as between the PBS and the editing template, and the relative positions as among elements of a PEgRNA are determined by the 5 ⁇ to 3 ⁇ order of the PEgRNA as a single molecule regardless of the position of sequences in the double stranded target DNA that may have complementarity or identity to elements of the PEgRNA.
  • the editing template is complementary or substantially complementary to a sequence on the PAM strand that is immediately downstream of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions.
  • the endogenous, e.g., genomic, sequence that is complementary or substantially complementary to the editing template, except for the one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit may be referred to as an “editing target sequence”.
  • the editing template has identity or substantial identity to a sequence on the target strand that is complementary to, or having the same position in the genome as, the editing target sequence, except for one or more one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the editing template encodes a single stranded DNA, wherein the single stranded DNA has identity or substantial identity to the editing target sequence except for one or more insertions, deletions, or substitutions at the positions of the one or more intended nucleotide edits.
  • the editing template may encode the wild-type or non-disease associated gene sequence (or its complement if the edit strand is the antisense strand of a gene).
  • the editing template may encode the wild-type or non-disease associated protein, but contain one or more synonymous mutations relative to the wild-type or non-disease associated protein coding region.
  • Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer).
  • a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene.
  • the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site.
  • a primer binding site (PBS) of the PEgRNA anneals with a free 3 ⁇ end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3 ⁇ end as a primer.
  • a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized.
  • the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence.
  • the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template.
  • the endogenous, e.g., genomic, sequence that is partially complementary to the editing template may be referred to as an “editing target sequence”.
  • the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template.
  • prime editing may comprise programmable editing of a target DNA using one or more prime editors each complexed with a PEgRNA (“dual prime editing”).
  • Dual prime editing refers to programmable editing of a double-stranded target DNA using two or more PEgRNAs, each of which is complexed with a prime editor for incorporating one or more intended nucleotide edits into the double-stranded target DNA.
  • dual prime editing incorporates one or more intended nucleotide edits into a double-stranded target DNA through excision of an endogenous DNA segment and/or replacement of the endogenous DNA segment with newly synthesized DNA via target-primed DNA synthesis.
  • dual prime editing may be used to edit a target DNA that is or is part of a target gene.
  • the target gene is a disease-associated gene.
  • the target gene is a monogenic disease- associated gene.
  • the target gene is a polygenic disease-associated gene.
  • the target gene is mutated compared to a wild-type sequence of the same gene and may cause or be associated with a disease or disorder in a subject.
  • the mutated target gene causes a disease or a disorder in a human subject.
  • dual prime editing involves using two different PEgRNAs each complexed with a prime editor, wherein each of the two PEgRNAs comprises a spacer complementary or substantially complementary to a separate search target sequence.
  • each of the two PEgRNAs anneals with a separate search target sequence through its spacer. Accordingly, references to a “PAM strand”, a “non-PAM strand”, a “target strand’, a “non- WSGR Docket No.59761-772601 target strand”, an “edit strand” or a “non-edit strand” are relative in the context of a specific PEgRNA, e.g., one of the two PEgRNAs in dual prime editing.
  • dual prime editing involves two PEgRNAs, different from one another, each complexed with a prime editor.
  • each of the two PEgRNAs comprises a region of complementarity to a distinct search target sequence of the target DNA, wherein the two distinct search target sequences are on the two complementary strands of the target DNA.
  • region region
  • portion and “segment” are used interchangeably to refer to a proportion of a molecule, for example, a polynucleotide or a polypeptide.
  • a region of a polynucleotide may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the polynucleotide.
  • the two PEgRNAs each can direct a prime editor to initiate the prime editing process on the two complementary strands of the target DNA.
  • dual prime editing involves two PEgRNAs each complexed with a prime editor.
  • a first PEgRNA comprises a first spacer complementary to a first search target sequence on a first strand of a double-stranded target DNA, e.g., a double-stranded target gene.
  • the first strand of the double-stranded target DNA may be referred to as a first target strand, and the complementary strand referred to as the first PAM strand.
  • a second PEgRNA comprises a second spacer complementary to a second search target sequence on a second strand of the double-stranded target DNA.
  • the first strand and the second strand of the double-stranded target DNA are complementary to each other. Accordingly, in some embodiments, the second PEgRNA and the first PEgRNA bind opposite strands of the double-stranded target DNA.
  • the second strand of the double-stranded target DNA may be referred to as a second target strand, and the complementary strand referred to as the second PAM strand.
  • the first target strand is the same strand as the second PAM strand of the double-stranded target DNA.
  • the second target strand is the same strand as the first PAM strand of the double-stranded target DNA.
  • the first PEgRNA anneals with the first target strand of the double- stranded target DNA, through the first spacer of the first PEgRNA.
  • the first PEgRNA complexes with and directs a first prime editor to bind the double-stranded target DNA at the position corresponding to the first search target sequence.
  • the second PEgRNA anneals with the second search target sequence on the second target strand of the double- stranded target DNA, through a second spacer of the second PEgRNA.
  • the second PEgRNA complexes with and directs a second prime editor to bind the double-stranded target DNA at the position corresponding to the second search target sequence.
  • the WSGR Docket No.59761-772601 first prime editor and the second prime editor are the same. In some embodiments, the first prime editor and the second prime editor are different.
  • the first search target sequence recognized by the spacer of the first PEgRNA and the second search target sequence recognized by the spacer of the second PEgRNA have a region of complementarity to each other. In some embodiments, the region of complementarity is 2 to 20 nucleotides in length.
  • the region of complementarity is 5 to 15 nucleotides in length.
  • the first search target sequence recognized by the spacer of the first PEgRNA and the second search target sequence recognized by the spacer of the second PEgRNA do not have a region of complementarity to each other.
  • the positions of the first and second search target sequences relative to each other may be determined by their positions in the double-stranded target DNA prior to editing. In some embodiments, the positions of the first and second search target sequences relative to each other may be determined by their positions in a reference double-stranded target DNA.
  • the first search target sequence is upstream of the second search target sequence.
  • the first search target sequence is downstream of the second search target sequence.
  • the 5’ end of the first search target sequence is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 base pairs upstream of the 5’ end of the second search target sequence.
  • the 5’ end of the first search target sequence is 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs upstream of the 5’ end of the second search target sequence.
  • the 5’ end of the first search target sequence is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more base pairs upstream of the 5’ end of the second search target sequence.
  • the 3’ end of the first search target sequence is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 base pairs downstream of the 3’ end of the second search target sequence.
  • the 3’ end of the first search target sequence is 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs WSGR Docket No.59761-772601 downstream of the 3’ end of the second search target sequence.
  • the 3’ end of the first search target sequence is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more base pairs downstream of the 3’ end of the second search target sequence.
  • the bound first prime editor generates a first nick on the first PAM strand of the double-stranded target DNA.
  • a first PEgRNA comprises a first primer binding site (PBS), also referred to herein as “primer binding site sequence”, that is complementary to the sequence of the first PAM strand of the double-stranded target DNA that is immediately upstream of the first nick site, and can anneal with the sequence of the first strand at a free 3’ end formed at the first nick site.
  • PBS primer binding site
  • a first PEgRNA comprises a first primer binding site (PBS) that anneals to a free 3’ end formed at the first nick site and the first prime editor initiates DNA synthesis from the nick site, using the free 3’ end as a primer.
  • the first prime editor generates a first newly synthesized single-stranded DNA encoded by a first editing template of the first PEgRNA.
  • the bound second prime editor generates a second nick on the second PAM strand of the double-stranded target DNA.
  • the double-stranded target DNA e.g., a target gene, comprises a double-stranded DNA sequence between the first nick generated by the first prime editor on the second target strand (also referred to as the first PAM strand) and the second nick generated by the second prime editor on the first target strand (also referred to as the second PAM strand), which may be referred to as an inter-nick duplex (IND).
  • IND inter-nick duplex
  • the two strands of an IND are completely complementary to each other. In some embodiments, the two strands of an IND are partially complementary to each other. In some embodiments, the IND is subsequently excised from the double-stranded target DNA, e.g., the target gene. [0267] In some embodiments, the IND is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs in length. In some embodiments, the IND is up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, or up to 50 base pairs in length.
  • the IND is 1-3000, 1-2500, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 base pairs in length.
  • the IND is 500- 3000, 500-2500, 500-2000, 500-1500, 500-1000, 500-900, 500-800, 500-700, or 500-600 base pairs in length.
  • the IND is 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30-50, 50- 200, 50-150, 50-100, 50-75, 75-100, 75-150, 75-200, 75-250, 75-300 base pairs in length.
  • the IND is 1-3, 1-6, 1-9, 1-12, 1-15, 1-18, 1-21, 1-24, 1-27, 1-30, 1-36, 1-45, 1-60, 1- 72, 1-90, 3-6, 3-9, 3-12, 3-15, 3-18, 3-21, 3-24, 3-27, 3-30, 3-36, 3-45, 3-60, 3-72, 3-90, 6-9, 6-12, 6- 15, 6-18, 6-21, 6-24, 6-27, 6-30, 6-36, 6-45, 6-60, 6-72, 6-90, 9-12, 9-15, 9-18, 9-21, 9-24, 9-27, 9-30, 9-36, 9-45, 9-60, 9-72, 9-90, 12-15, 12-18, 12-21, 12-24, 12-27, 12-30, 12-36, 12-45, 12-60, 12-72, 12-90, 15-18, 15-21, 15-24, 15-27, 15-30, 15-36, 15-45, 15-60, 15-72, 15-90, 18-21, 18-24, 18-27, 18-
  • the IND is 1-3000, 1-2500, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1- 200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 500-3000, 500-2500, 500-2000, 500-1500, 500-1000, 500-900, 500-800, 500-700, 500-600, 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30- 50, 50-200, 50-150, 50-100, 50-75, 75-100, 75-150, 75-200, 75-250, or 75-300 base pairs in length.
  • the IND is 1-3, 1-6, 1-9, 1-12, 1-15, 1-18, 1-21, 1-24, 1-27, 1-30, 1-36, 1-45, 1- 60, 1-72, 1-90, 3-6, 3-9, 3-12, 3-15, 3-18, 3-21, 3-24, 3-27, 3-30, 3-36, 3-45, 3-60, 3-72, 3-90, 6-9, 6- 12, 6-15, 6-18, 6-21, 6-24, 6-27, 6-30, 6-36, 6-45, 6-60, 6-72, 6-90, 9-12, 9-15, 9-18, 9-21, 9-24, 9-27, 9-30, 9-36, 9-45, 9-60, 9-72, 9-90, 12-15, 12-18, 12-21, 12-24, 12-27, 12-30, 12-36, 12-45, 12-60, 12- 72, 12-90, 15-18, 15-21, 15-24, 15-27, 15-30, 15-36, 15-45, 15-60, 15-72, 15-90, 18-21, 18-24, 18-27, 18-30
  • the double-stranded target DNA is a double-stranded target gene or a part of a double-stranded target gene
  • the IND comprises a part of a coding sequence of the target gene.
  • the IND comprises a part of a non-coding sequence of the target gene.
  • the IND comprises a part of an exon.
  • the IND comprises an entire exon.
  • the IND comprises a part of an intron.
  • the IND comprises an entire intron.
  • the IND comprises a 3’ UTR sequence of the target gene.
  • the IND comprises a 5’ UTR sequence of the target gene.
  • the IND comprises a whole or a part of an ORF of the target gene. In some embodiments, the IND comprises both coding and non-coding sequences of the target gene. In some embodiments, the IND comprises both intron and exon sequences of the target gene. For example, in some embodiments, the IND comprises the sequence of an exon flanked by an intronic sequence at the 5’ end, the 3’ end, or both ends. In some embodiments, the IND comprises one or more exons and intervening introns. In some embodiments, the IND comprises two or more exons and intervening introns.
  • the IND comprises all of the coding regions of a target gene, regulatory sequences of a target gene, or the entire target gene comprising its exons, introns and regulatory sequences.
  • the double-stranded DNA comprises a gene or a part of a gene, and the IND comprises one or more mutations compared to a wild-type reference sequence of the same gene.
  • the one or more mutations are associated with a disease.
  • a first PEgRNA comprises a first primer binding site (PBS) that is complementary to a free 3’ end of the second strand of the double-stranded target DNA formed at the first nick site.
  • the first PBS anneals with the free 3’ end formed at the first nick site, and the first prime editor initiates DNA synthesis from the first nick site, using the free 3’ end at WSGR Docket No.59761-772601 the first nick site as a primer.
  • the first prime editor synthesizes a first new single-stranded DNA encoded by the first editing template of the first PEgRNA.
  • the second PEgRNA comprises a second PBS that is complementary to a free 3’ end of the first strand of the double-stranded target DNA formed at the second nick site.
  • the second PBS anneals with the free 3’ end formed at the second nick site, and the second prime editor initiates DNA synthesis from the nick site, using the free 3’ end at the second nick site as a primer.
  • the second prime editor synthesizes a second newly synthesized single-stranded DNA encoded by a second editing template of the second PEgRNA.
  • the sequence of the first newly synthesized single-stranded DNA encoded by the first editing template and/or the sequence of the second newly synthesized single-stranded DNA encoded by the second editing template is incorporated into the double-stranded target DNA, e.g., a target gene, thereby incorporating one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene.
  • a “nucleotide edit” or an “intended nucleotide edit” refers to a specified edit of a double-stranded target DNA.
  • a nucleotide edit or intended nucleotide edit refers to a (i) deletion of one or more contiguous nucleotides at one specific position, (ii) insertion of one or more contiguous nucleotides at one specific position, (iii) substitution of one or more contiguous nucleotides, or (iv) a combination of contiguous nucleotide substitutions, insertions and/or deletions of two or more contiguous nucleotides at one specific position, or other alterations at one specific position to be incorporated into the sequence of the double-stranded target DNA.
  • An intended nucleotide edit may refer to the edit on an editing template (e.g., a first editing template or a second editing template) as compared to the sequence of the double-stranded target gene, or may refer to the edit encoded by an editing template in the newly synthesized single-stranded DNA that is incorporated in the double-stranded target DNA, e.g., the TRAC gene, as compared to endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • an editing template e.g., a first editing template or a second editing template
  • an intended nucleotide edit may also refer to the edit that results from incorporation of the newly synthesized DNA encoded by an editing template, or incorporation of the two newly synthesized single-stranded DNA encoded by each of the first PEgRNA and the second PEgRNA in dual prime editing.
  • the sequence of the first newly synthesized single-stranded DNA and/or the sequence of the second newly synthesized single-stranded DNA are incorporated into the double-stranded target DNA, e.g., the target gene.
  • the first and/or the second newly synthesized single-stranded DNAs comprises one or more intended nucleotide edits compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene, which are incorporated in the double-stranded target DNA, e.g., the target gene.
  • the sequence of the first newly synthesized single-stranded DNA encoded by the first editing template is incorporated in the double-stranded target DNA, e.g., the target gene, thereby incorporating one or WSGR Docket No.59761-772601 more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene.
  • the sequence of the second newly synthesized single-stranded DNA encoded by the second editing template is incorporated in the double-stranded target DNA, e.g., the target gene, thereby incorporating one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene.
  • the sequence of the first newly synthesized single-stranded DNA encoded by the first editing template and the sequence of the second newly synthesized single- stranded DNA encoded by the second editing template are incorporated in the double-stranded target DNA, e.g., the target gene, thereby incorporating one or more intended nucleotide edits in the double- stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises an insertion, deletion, nucleotide substitution, inversion, or any combination thereof compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide substitutions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene. In some embodiments, the intended nucleotide edit comprises up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, or up to 50 nucleotide substitutions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 nucleotide substitutions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene. In some embodiments, the intended nucleotide edit comprises 3-50, 3-40, 3-30, 3-25, 3- 20, 3-15, 3-10, or 3-5 nucleotide substitutions compared to the endogenous sequence of the double- stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, or 5-10 nucleotide substitutions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide insertions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, or up to 50 nucleotide insertions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises single nucleotide insertions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sites in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises nucleotide insertions of greater than one nucleotide at each site in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • site refers to a specific position in the sequence of a target DNA, e.g., a target gene.
  • the specific position in the sequence of the double-stranded target DNA e.g., a target WSGR Docket No.59761-772601 gene, can be referred to by specific positions in a reference sequence, e.g., a wild-type gene sequence.
  • a nucleotide insertion at position x refers to insertion of one or more nucleotides between position x and position x+1 as set forth by numbering in a reference sequence.
  • a nucleotide deletion at position x refers to deletion of the specific nucleotide at position x as set forth by numbering in a reference sequence.
  • a nucleotide deletion of positions x to x+n refers to deletion of the specific nucleotides starting at nucleotide x to nucleotide x+n, including nucleotide x and nucleotide x+n, as set forth by numbering in a reference sequence.
  • a nucleotide inversion of positions x to x+n refers to inversion of the specific nucleotides starting at nucleotide x to nucleotide x+n, including nucleotide x and nucleotide x+n, as set forth by numbering in a reference sequence.
  • the intended nucleotide edit comprises nucleotide insertions of greater than one nucleotide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sites in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1-3000, 1- 2500, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 nucleotide insertions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 500-3000, 500-2500, 500-2000, 500-1500, 500-1000, 500-900, 500-800, 500-700, or 500-600 nucleotide insertions compared to the endogenous sequence of the double- stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30-50, 50-200, 50-150, 50-100, 50-75, 75- 100, 75-150, 75-200, 75-250, or 75-300 nucleotide insertions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises nucleotide insertions of 1-3000, 1-2500, 1-2000, 1-1500, 1-1000, 1-900, 1- 800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 500-3000, 500-2500, 500-2000, 500-1500, 500-1000, 500-900, 500-800, 500-700, 500-600, 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30-50, 50-200, 50-150, 50-100, 50-75, 75-100, 75-150, 75- 200, 75-250, or 75-300 nucleotides at each site in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide deletions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, or up to 50 nucleotide deletions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises single nucleotide deletions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sites in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target WSGR Docket No.59761-772601 DNA, e.g., the target gene.
  • the intended nucleotide edit comprises nucleotide deletions of greater than one nucleotide at each site in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises nucleotide deletions of greater than one nucleotide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sites in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1-3000, 1- 2500, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 nucleotide deletions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 500-3000, 500-2500, 500-2000, 500-1500, 500-1000, 500-900, 500-800, 500-700, or 500-600 nucleotide deletions compared to the endogenous sequence of the double- stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30-50, 50-200, 50-150, 50-100, 50-75, 75- 100, 75-150, 75-200, 75-250, 75-300 nucleotide deletions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises 1-3, 1-6, 1-9, 1-12, 1-15, 1-18, 1-21, 1-24, 1-27, 1-30, 1-36, 1-45, 1-60, 1-72, 1-90, 3-6, 3-9, 3-12, 3-15, 3-18, 3-21, 3-24, 3-27, 3-30, 3-36, 3-45, 3-60, 3-72, 3-90, 6-9, 6-12, 6-15, 6-18, 6-21, 6-24, 6-27, 6-30, 6-36, 6-45, 6-60, 6-72, 6- 90, 9-12, 9-15, 9-18, 9-21, 9-24, 9-27, 9-30, 9-36, 9-45, 9-60, 9-72, 9-90, 12-15, 12-18, 12-21, 12-24, 12-27, 12-30, 12-36, 12-45, 12-60, 12-72, 12-90, 15-18, 15-21, 15-24, 15-27, 15-30, 15-36, 15-45, 15- 60, 15-72, 15-90, 18
  • the intended nucleotide edit comprises nucleotide deletions of 1-3000, 1-2500, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1- 100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 500-3000, 500-2500, 500-2000, 500-1500, 500- 1000, 500-900, 500-800, 500-700, 500-600, 30-300, 30-250, 30-200, 30-150, 30-100, 30-75, 30-50, 50-200, 50-150, 50-100, 50-75, 75-100, 75-150, 75-200, 75-250, or 75-300 nucleotides at each site in the double-stranded target DNA compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises nucleotide deletions of 1-3, 1-6, 1-9, 1-12, 1-15, 1-18, 1-21, 1-24, 1-27, 1-30, 1-36, 1-45, 1-60, 1-72, 1-90, 3-6, 3-9, 3-12, 3-15, 3-18, 3-21, 3-24, 3-27, 3-30, 3-36, 3-45, 3-60, 3-72, 3-90, 6-9, 6-12, 6-15, 6-18, 6-21, 6-24, 6-27, 6-30, 6-36, 6-45, 6-60, 6-72, 6-90, 9-12, 9-15, 9-18, 9-21, 9-24, 9-27, 9-30, 9-36, 9-45, 9- 60, 9-72, 9-90, 12-15, 12-18, 12-21, 12-24, 12-27, 12-30, 12-36, 12-45, 12-60, 12-72, 12-90, 15-18, 15-21, 15-24, 15-27, 15-30, 15-36, 15-45, 15-60, 15-72, 15-90, 15
  • the intended nucleotide edits are in consecutive or contiguous nucleotides in the double-stranded target DNA sequence compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edits e.g., nucleotide substitutions, insertions, or deletions are in non-consecutive or non-contiguous nucleotides in the double-stranded target DNA sequence compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises an inversion as compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • a segment of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300 or more nucleotides of the endogenous sequence of the double-stranded target DNA is inverted.
  • a segment of 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 nucleotides of the endogenous sequence of the double-stranded target DNA is inverted.
  • a segment of 3-50, 3-40, 3-30, 3-25, 3-20, 3-15, 3-10, 3-5, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, or 5-10 nucleotides of the endogenous sequence of the double-stranded target DNA is inverted.
  • the intended nucleotide edit comprises more than one nucleotide edit in the double-stranded target DNA sequence compared to the endogenous sequence of the double- stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises a combination of one or more of nucleotide substitutions, one or more of nucleotide insertions, one or more of nucleotide deletions and one or more of nucleotide inversions compared to the endogenous sequence of the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit comprises one or more nucleotide substitutions and one or more nucleotide insertions.
  • the intended nucleotide edit comprises one or more nucleotide substitutions and one or more nucleotide deletions.
  • the intended nucleotide edit comprises one or more nucleotide substitutions and one or more nucleotide inversions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide insertions and one or more nucleotide deletions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide insertions and one or more nucleotide inversions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide deletions and one or more nucleotide inversions.
  • the intended nucleotide edit comprises one or more nucleotide substitutions, one or more nucleotide insertions and one or more nucleotide deletions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide substitutions, one or WSGR Docket No.59761-772601 more nucleotide insertions and one or more nucleotide inversions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide substitutions, one or more nucleotide deletions and one or more nucleotide inversions.
  • the intended nucleotide edit comprises one or more nucleotide insertions, one or more nucleotide deletions and one or more nucleotide inversions. In some embodiments, the intended nucleotide edit comprises one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions and one or more nucleotide inversions. [0281] In some embodiments, the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA have a region of complementarity to each other.
  • the first newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, adjacent to or near a nick site. In some embodiments, the first newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, on the first strand adjacent to the second nick site.
  • the first newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double- stranded target DNA, e.g., the target gene, on the first strand adjacent to and downstream of the second nick site. In some embodiments, the first newly synthesized single-stranded DNA has a region of identity to an endogenous sequence of the double-stranded target on the second strand adjacent to and downstream of the first nick site. In some embodiments, the first newly synthesized single- stranded DNA has a region of identity to an endogenous sequence of the double-stranded target on the second strand adjacent to and upstream of the second nick site.
  • the second newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, adjacent to or near a nick site. In some embodiments, the second newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, on the second strand adjacent to the first nick site.
  • the second newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, on the second strand adjacent to and downstream of the first nick site. In some embodiments, the second newly synthesized single- stranded DNA has a region of identity to an endogenous sequence of the double-stranded target on the first strand adjacent to and upstream of the second nick site. [0283] As used herein, reference for positioning in a chromosome or a double-stranded polynucleotide, e.g., a double-stranded target DNA, includes the position on either strand of the two strands, unless otherwise specified.
  • a position of a first nick site may be used refer to the first nick site on the first edit strand and/or the corresponding position on the second edit strand.
  • WSGR Docket No.59761-772601 By “upstream” and “downstream” it is intended to define relative positions of at least two regions or sequences in a nucleic acid molecule oriented in a 5 ⁇ -to-3 ⁇ direction.
  • a first sequence is upstream of a second sequence in a DNA molecule where the first sequence is positioned 5’ to the second sequence. Accordingly, the second sequence is downstream, that is, 3’, of the first sequence.
  • the 5’ to 3’ direction is based on a reference strand that is the protein encoding strand (also referred to as the sense strand) of the double-stranded target DNA, e.g., the HTT gene, regardless of whether the sequence is in a translated region.
  • the reference strand is the first edit strand (i.e. the second strand as shown in FIG.4A).
  • sequence defined as the upstream (or the 5’) sequence and the sequence defined as the downstream (or the 3’) sequence are based on the position of the sequence on the sense strand (e.g., the first edit strand as exemplified in Fig.4A) compared to the position of the complementary sequence of the sequence on the antisense strand.
  • each of the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, adjacent to or near a nick site.
  • each of the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA has a region of identity to an endogenous sequence of the double- stranded target DNA, e.g., the target gene, adjacent to or near a nick site.
  • the first newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, on the first strand adjacent to and downstream of the second nick site
  • the second newly synthesized single-stranded DNA has a region of complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the target gene, on the second strand adjacent to and upstream of the first nick site.
  • the first newly synthesized single-stranded DNA has a region of identity to an endogenous sequence of the double-stranded target DNA on the second strand adjacent to and downstream of the first nick site and/or a region of identity to an endogenous sequence of the double-stranded target DNA on the second strand adjacent to and upstream of the second nick site
  • the second newly synthesized single-stranded DNA has a region of identity to an endogenous sequence of the double-stranded target DNA on the first strand adjacent to and downstream of the first nick site and/or a region of identity to an endogenous sequence of the double-stranded target DNA on the second strand adjacent to and upstream of the second nick site.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second WSGR Docket No.59761-772601 editing template have a region of complementarity to each other.
  • the complementary region between the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA may be referred to as an overlap duplex (OD).
  • OD overlap duplex
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template are complementary or substantially complementary to each other.
  • the OD is incorporated in the double-stranded target DNA, e.g., the target gene, thereby incorporating one or more intended nucleotide edits encoded by the first editing template and the second editing template into the double-stranded target DNA, e.g., the target gene.
  • the OD replaces all or a portion of the IND, thereby incorporating one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene.
  • the IND is excised or degraded, and the OD is incorporated at the place of the IND excision, followed by ligation of the nicks on both strands of the double-stranded target DNA, e.g., the target gene, thereby incorporating the one or more intended nucleotide edits in the double-stranded target DNA.
  • the sequence of the OD comprises partial identity compared to the sequence of the IND. In some embodiments, the sequence of the OD comprises no identity compared to the sequence of the IND. In some embodiments, the sequence of the OD comprises a sequence exogenous to the double-stranded target DNA.
  • incorporation of the OD does not alter the reading frame of the double-stranded target DNA.
  • the first editing template and the second editing template comprise a region of complementarity or substantial complementarity to each other, and do not have complementarity to either strand of the double-stranded target DNA, e.g., the target gene.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template can anneal to each other to form an OD that does not have nucleotide sequence identity with the endogenous sequence of double-stranded target DNA, e.g., the target gene.
  • the sequence of the OD comprises a sequence exogenous to the double-stranded target DNA, e.g., the target gene. In some embodiments, the sequence of the OD consists of a sequence exogenous to the double-stranded target DNA, e.g., the target gene. In some embodiments, the IND is excised, and the OD is incorporated at the place of the IND excision, followed by ligation of the nicks on both strands of the target DNA, thereby incorporating the sequence of the OD in the double- stranded target DNA.
  • the OD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more contiguous complementary or substantially complementary base pairs.
  • the OD comprises about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to WSGR Docket No.59761-772601 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 15 to 55, 15 to 60, 15 to 65, 15 to 70, 15 to 75, 15 to 80, 15 to 85, 15 to 90, 15 to 95, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140,
  • the OD comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous complementary or substantially complementary base pairs. In some embodiments, the OD comprise 30, 35, 40, 50, 60, 70, 80, 90, or 100 contiguous complementary or substantially complementary base pairs. In some embodiments, the OD comprise no greater than 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 contiguous complementary or substantially complementary base pairs. In some embodiments, the OD comprises a sufficient number of contiguous complementary base pairs to form a sufficiently stable duplex for replacement of the IND. In some embodiments, the OD comprises at least 10 contiguous complementary or substantially complementary base pairs. In some embodiments, the OD comprises at least 15 contiguous complementary or substantially complementary base pairs.
  • the OD comprises about 20 contiguous complementary or substantially complementary base pairs.
  • the OD replaces the IND of a target DNA, wherein the double- stranded target DNA is an entire target gene or is part of a target gene. In some embodiments, the OD replaces part of an exon or an entire exon, part of an intron or an entire intron, one or more exons and intervening introns, all of the coding regions of a target gene, regulatory sequences of a target gene, or the entire target gene comprising its exons, introns and regulatory sequences. In some embodiments, the OD comprises a region of identity to an endogenous sequence of the double-stranded target DNA.
  • the OD does not have sequence identity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the OD is exogenous to the double-stranded target DNA, e.g., the target gene.
  • WSGR Docket No.59761-772601 [0291]
  • the OD has a biological function or encodes a polypeptide having a biological function, or a portion thereof.
  • the OD comprises an expression cassette.
  • the OD comprises a nucleotide sequence that encodes an expression tag, for example, an affinity tag, a His tag, a V5 tag, or a FLAG tag.
  • the OD comprises a nucleotide sequence that encodes a His tag. In some embodiments, the OD comprises a nucleotide sequence that encodes a FLAG tag. In some embodiments, the OD comprises a nucleotide sequence that encodes an attB or an attP sequence. In some embodiments, the OD comprises a nucleotide sequence that encodes a reporter protein, for example, a green fluorescence protein, a blue fluorescence protein, a cyan fluorescence protein, a yellow fluorescence protein, an auto fluorescent protein, or a luciferase. In some embodiments, the OD comprises a recognition site of an enzyme, for example, a recombinase recognition sequence.
  • the OD comprises nucleotide sequence that encodes a selectable marker, for example, an antibiotic resistance marker.
  • the OD comprises a regulatory sequence, for example, a promoter, an enhancer, or an insulator.
  • the OD comprises a trackable sequence, for example, a barcode.
  • replacement of the IND by the OD decreases or abolishes the expression or the function of the target gene (e.g., TRAC gene).
  • replacement of the IND by the OD results in disruption of the target DNA (e.g., a TRAC gene) and insertion of one or more recombinase recognition sequences encoded by the OD.
  • the target gene is a disease-associated gene. In some embodiments, the target gene is a monogenic disease-associated gene. In some embodiments, the target gene is a polygenic disease-associated gene. In some embodiments, the target gene is a disease-associated gene containing one or more disease-causing mutations, wherein replacement of the IND by the OD corrects the mutations, thereby restoring or partially restoring the function of the target gene. In some embodiments, the disease-associated gene containing one or more disease-causing mutations is in a human subject in need of treatment.
  • the target gene is a mutated gene causing a disease or disorder in a human subject, wherein replacement of the IND by the OD corrects the mutated gene, thereby restoring or partially restoring the function of the target gene.
  • the target gene is a disease-associated gene containing one or more disease-causing mutations, wherein replacement of the IND by the OD modifies the target gene to restore or partially restore the function of the target gene.
  • the disease-associated gene containing one or more disease-causing mutations is in a human subject in need of treatment.
  • the target gene is a mutated gene causing a disease or disorder in a human subject, wherein replacement of the IND by the OD modifies the mutated gene to restore or partially restore the function of the target gene.
  • the target gene is a wildtype gene, e.g., a wildtype TRAC.
  • replacement of the IND by the OD modifies the target gene to decrease expression or function of the target gene, a mRNA or a protein encoded by the target gene.
  • the target gene is a TRAC gene.
  • replacement of the IND by the OD modifies the TRAC gene to decrease a function of WSGR Docket No.59761-772601 the TRAC gene, TRAC mRNA and/or TRAC protein encoded by the TRAC gene.
  • replacement of the IND by the OD results in disruption of the target gene (e.g., a TRAC gene) and insertion of one or more exogenous sequences, e.g., one or more recombinase recognition sequences into the gene.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity with each other, and can anneal with each other to form an OD.
  • the first newly synthesized single-stranded DNA encoded by the first editing template further comprises a region that does not have complementarity with the second newly synthesized single-stranded DNA encoded by the second editing template (see exemplary schematic in FIG.4B).
  • the second newly synthesized single- stranded DNA encoded by the second editing template further comprises a region that does not have complementarity with the first newly synthesized single-stranded DNA encoded by the first editing template. Accordingly, in some embodiments, the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template can anneal to each other through the partially complementary sequences to form an OD that is linked to a 5’ overhang and/or a 3’overhang.
  • the IND is removed, the OD, along with the 5’ overhang and/or the 3’ overhang, is incorporated at the place of the IND excision in the double-stranded target DNA, e.g., the target gene.
  • the gaps corresponding to the positions of the 5’ overhang and/or the 3’ overhangs are filled and ligated, thereby incorporating the one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene.
  • the IND is replaced by the sequence of (A+C), (B+C), or (A+B+C), wherein A is the region, and its complementary strand, of the first newly synthesized single-stranded DNA that is not complementary to the second newly synthesized single-stranded DNA, wherein B is the region, and its complementary strand, of the second newly synthesized single- stranded DNA that is not complementary to the first newly synthesized single-stranded DNA, and wherein C is the OD.
  • the double-stranded sequence of (A+C), (B+C), or (A+B+C) that replaces the IND may be referred to as the “replacement duplex (RD)”.
  • the RD comprises the OD.
  • the first editing template and the second editing template are substantially complementary to each other.
  • the OD comprises the entirety or substantially the entirety of the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template.
  • the RD consists of the OD. In some embodiments, as exemplified in FIG.
  • the RD comprises the OD, the non-complementary region of the first newly WSGR Docket No.59761-772601 synthesized DNA compared to the second newly synthesized DNA and complement thereof, and/or the non-complementary region of the second newly synthesized DNA compared to the first newly synthesized DNA and complement thereof.
  • the RD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more base pairs.
  • the RD comprises about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 17
  • the RD comprise 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs.
  • the RD comprise at least 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs.
  • the RD comprise no greater than 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs.
  • the RD replaces the IND of a target DNA, wherein the IND is an entire target gene or is part of a target gene.
  • the RD replaces part of an exon or an entire exon, part of an intron or an entire intron, one or more exons and intervening introns, all of the coding regions of a target gene, regulatory sequences of a target gene, or the entire target gene comprising its exons, introns and regulatory sequences, thereby incorporating the one or more intended nucleotide edits compared to the endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the RD comprises a region of identity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the RD does not have sequence identity to an endogenous sequence of the double-stranded target DNA.
  • the RD is exogenous to the double-stranded target DNA, e.g., the target gene.
  • the intended nucleotide edit(s) comprises replacement of an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene, in its entirety, by the sequence of the RD.
  • the RD or the OD may comprise a recombinase recognition sequences (RSSs), e.g., a RSS recognized by a Bxbl recombinase, a Cre recombinase, a Pa01 recombinase, a Si74 recoimbinase, a No67 recombinase, a Kp03 recombinase, a Nm60 recombinase, a BceINTa recombinase, a NcytINTd recombinase, a SscINTd recombinase, a SacINTd recombinase, or a recombinase recognition site corresponding to any recombinase disclosed herein.
  • RSSs recombinase recognition sequences
  • the RD or the OD may comprise one, two, or more recombinase recognition sites corresponding to a recombinase.
  • Replacement of the IND by the RD or the OD comprising one or more recombinase sequences with dual prime editing may result in insertion of the one or more recombinase sequences into the target gene, e.g., a TRAC gene.
  • the target gene e.g., a TRAC gene.
  • they can be used as landing sites for a recombinase-mediated reaction between the RSSs.
  • a single RSS inserted into a target gene can be used for integration of an exogenous DNA donor sequence via recombination between the inserted RSS and a second RSS within the exogenous supplied DNA donor
  • a target gene e.g., a TRAC gene
  • two recombinase sites are inserted in adjacent regions of DNA, depending on the orientation of the recombinase sites, these can be used for recombinase-mediated excision or inversion of the intervening sequence, or for recombinase- mediated cassette exchange with exogenous DNA for cargo integration.
  • the RD has a biological function or encodes a polypeptide having a biological function.
  • the RD comprises an expression cassette.
  • the RD comprises a nucleotide sequence that encodes an expression tag, for example, an affinity tag, a His tag, a V5 tag, or a FLAG tag. In some embodiments, the RD comprises a nucleotide sequence that encodes a His tag. In some embodiments, the RD comprises a nucleotide sequence that encodes a FLAG tag. In some embodiments, the RD comprises a nucleotide sequence that encodes an attB or an attP sequence.
  • the RD comprises a nucleotide sequence that encodes a reporter protein, for example, a green fluorescence protein, a blue fluorescence protein, a cyan fluorescence protein, a yellow fluorescence protein, an auto fluorescent protein, or a luciferase.
  • the RD comprises a recognition site of an enzyme, for example, a recombinase recognition sequence.
  • the RD comprises a nucleotide sequence that encodes a selectable marker, for example, an antibiotic resistance marker.
  • the RD comprises a regulatory sequence, for example, a promoter, an enhancer, or an insulator.
  • the RD comprises a trackable sequence, for example, a barcode.
  • replacement of the IND by the RD restores or partially restores the function of the target gene.
  • replacement of the IND by the RD decreases or abolishes the function or expression of the target gene.
  • the target gene is a TRAC gene.
  • replacement of the IND by the RD decreases the function of the TRAC gene, TRAC mRNA and/or TRAC protein.
  • the target gene is a disease-associated gene.
  • the target gene is a monogenic disease-associated gene.
  • the target gene is a polygenic disease-associated gene.
  • the target gene is a disease-associated gene containing one or more disease-causing mutations, wherein replacement of the IND by the RD corrects the mutations, thereby restoring or partially restoring the function of the target gene.
  • the disease-associated gene containing one or more disease-causing mutations is in a human subject in need of treatment.
  • the target WSGR Docket No.59761-772601 gene is a mutated gene causing a disease or disorder in a human subject, wherein replacement of the IND by the RD corrects the mutated gene, thereby restoring or partially restoring the function of the target gene.
  • the target gene is a disease-associated gene containing one or more disease-causing mutations, wherein replacement of the IND by the RD modifies the target gene to restore or partially restore the function of the target gene.
  • the disease- associated gene containing one or more disease-causing mutations is in a human subject in need of treatment.
  • the target gene is a mutated gene causing a disease or disorder in a human subject, wherein replacement of the IND by the RD modifies the mutated gene to restore or partially restore the function of the target gene.
  • the first editing template and the second editing template are partially complementary to each other.
  • the first editing template is partially complementary to the second editing template when the first and the second editing templates have complementary or substantially complementary region(s) over part of the length of both editing templates.
  • the partially complementary region(s) in the first editing template and the second editing template can be in any position within the first editing template and the second editing template.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template are partially complementary to each other, at any position within the first newly synthesized single- stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template.
  • the first newly synthesized single-stranded DNA comprises a region of complementarity to the second newly synthesized single- stranded DNA, at or near the 3’ end of the first newly synthesized single-stranded DNA. In some embodiments, the first newly synthesized single-stranded DNA comprises a region of complementarity to the second newly synthesized single-stranded DNA, at or near the 5’ end of the first newly synthesized single-stranded DNA. In some embodiments, the first newly synthesized single-stranded DNA comprises a region of complementarity to the second newly synthesized single- stranded DNA, in the middle of the first newly synthesized single-stranded DNA.
  • the second newly synthesized single-stranded DNA comprises a region of complementarity to the first newly synthesized single-stranded DNA, at or near the 3’ end of the second newly synthesized single-stranded DNA. In some embodiments, the second newly synthesized single-stranded DNA comprises a region of complementarity to the first newly synthesized single-stranded DNA, at or near the 5’ end of the second newly synthesized single- stranded DNA. In some embodiments, the second newly synthesized single-stranded DNA comprises a region of complementarity to the first newly synthesized single-stranded DNA, in the middle of the second newly synthesized single-stranded DNA.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA each comprises a region of complementarity to each other at the 3’ end of each of the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA.
  • the first editing template and the second editing template are of the same length. In some embodiments, the first editing template and the second editing template are of different lengths.
  • the first editing template comprises a region that has complementarity or substantial complementarity to the second editing template (the OD encoding region), and further comprises a region that does not have complementarity to the second editing template.
  • the first editing template comprises a region that has complementarity or substantial complementarity to the second editing template (the OD encoding region), wherein the region is flanked by one or more regions that do not have complementarity to the second editing template.
  • the entirety of the first editing template has complementarity or substantial complementarity to a region of the second editing template, wherein the second editing template comprises a region that does not have complementarity to the first editing template.
  • the first editing template comprises a region that does not have complementarity to the second editing template, wherein the region is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95, 10
  • the first editing template comprises a region that does not have complementarity to the second editing template, wherein the region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 or more nucleotides in length.
  • the second editing template comprises a region that has complementarity or substantial complementarity to the first editing template, and further comprises a region that does not have complementarity to the first editing template. In some embodiments, the second editing template comprises a region that has complementarity or substantial complementarity to the first editing template, and is flanked by one or more regions that do not have complementarity to the first editing template. The region(s) in the first editing template and the second editing template may have same or different lengths.
  • the entirety of the second editing template has complementarity or substantial complementarity to a region of the first editing template, wherein WSGR Docket No.59761-772601 the first editing template comprises a region that does not have complementarity to the second editing template.
  • the second editing template comprises a region that does not have complementarity to the first editing template, wherein the region is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95, 10
  • the second editing template comprises a region that does not have complementarity to the first editing template, wherein the region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 or more nucleotides in length.
  • the RD comprises a region (or a subset) of the sequence of the IND. In some embodiments, the RD consists of a region of the sequence of the IND. In some embodiments, the RD comprises one or more intended nucleotide edits compared to the IND. In some embodiments, the RD comprises a region(s) that has substantial sequence identity to the sequence of the IND, wherein the region(s) comprises one or more nucleotide edits compared to the sequence of the IND. For example, the RD may comprise a region that has substantial sequence identity to the sequence of the IND, wherein the region comprises one or more nucleotide substitutions, insertions, or deletions.
  • the RD comprises a region of the sequence of the IND, and further comprises a region that does not have sequence identity or complementary to the IND. In some embodiments, the RD comprises a region that has substantial identity to the sequence of the IND comprising one or more nucleotide edits, and further comprises a region that does not have sequence identity or complementary to the IND. In some embodiments, the region that does not have sequence identity or complementary to the IND has a biological function or encodes a polypeptide or a portion thereof having a biological function. In some embodiments, the RD comprises one or more intended nucleotide edits compared to the IND and encodes a polypeptide or a portion thereof.
  • the OD comprises a region (or a subset) of the sequence of the IND. In some embodiments, the OD consists of a region of the sequence of the IND. In some embodiments, the OD comprises one or more intended nucleotide edits compared to the IND. In some embodiments, the OD comprises a region(s) that has substantial sequence identity to the sequence of the IND, wherein the region(s) comprise one or more nucleotide edits compared to the sequence of the IND. For example, the OD may comprise a region that has substantial sequence identity to the sequence of the IND, wherein the region comprises one or more nucleotide substitutions, insertions, or deletions.
  • the OD comprises a region of the sequence of the IND, and further comprises a WSGR Docket No.59761-772601 region that does not have sequence identity or complementary to the IND.
  • the OD comprises a region that has substantial identity to the sequence of the IND comprising one or more nucleotide edits, and further comprises a region that does not have sequence identity or complementarity to the IND.
  • the region that does not have sequence identity or complementarity to the IND has a biological function or encodes a polypeptide or a portion thereof having a biological function.
  • the OD comprises one or more intended nucleotide edits compared to the IND and encodes a polypeptide or a portion thereof.
  • the first editing template comprises a region of identity to a sequence adjacent to the second nick site on the second PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND.
  • the second editing template comprises a region of identity to a sequence adjacent to the first nick site on the first PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region of complementarity to a sequence adjacent to the second nick site on the second PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence adjacent to the first nick site on the first PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND.
  • the first newly synthesized single-stranded DNA comprises a region of complementarity to a sequence immediately adjacent to the second nick site on the second PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence immediately adjacent to the first nick site on the first PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND (see, e.g., FIG.4F).
  • the first newly synthesized single-stranded DNA comprises a region of complementarity to a sequence adjacent to the second nick site on the second PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from the second nick site.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence adjacent to the first nick site on the first PAM strand of the double-stranded target DNA, wherein the sequence is outside the IND, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from the first nick site.
  • the first editing template and the second editing template each comprises a region of complementarity or substantial complementarity to each other.
  • the first editing template comprises a sequence that is exogenous to the double-stranded WSGR Docket No.59761-772601 target DNA.
  • the second editing template comprise a sequence that is exogenous to the double-stranded target DNA.
  • the sequence in the first editing template that is exogenous to the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence in the second editing template that is exogenous to the double-stranded target DNA.
  • the sequence in the first editing template that is exogenous to the double-stranded target DNA further comprises a region that is not complementary to the sequence in the second editing template that is exogenous to the double-stranded target DNA.
  • the sequence in the second editing template that is exogenous to the double- stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence in the first editing template that is exogenous to the double-stranded target DNA.
  • the sequence in the second editing template that is exogenous to the double-stranded target DNA further comprises a region that is not complementary to the sequence in the first editing template that is exogenous to the double-stranded target DNA.
  • the first editing template comprises a sequence exogenous to the double-stranded target DNA, wherein the sequence exogenous to the double-stranded target DNA comprises a polynucleotide sequence that encodes an expression tag, for example, an affinity tag, a His tag, a V5 tag, or a FLAG tag.
  • the second editing template comprises a sequence exogenous to the double-stranded target DNA, wherein the sequence exogenous to the double-stranded target DNA comprises a polynucleotide sequence that encodes an expression tag, for example, an affinity tag, a His tag, a V5 tag, or a FLAG tag.
  • the first editing template and/or the second editing template comprises a sequence exogenous to the double-stranded target DNA, wherein the exogenous sequence comprises a recombinase recognition sequence (RSS), e.g., an attB sequence.
  • RSS recombinase recognition sequence
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA each comprises a region of complementarity or substantial complementarity to each other.
  • the first newly synthesized single- stranded DNA comprise a sequence that is exogenous to the double-stranded target DNA.
  • the second newly synthesized single-stranded DNA comprise a sequence that is exogenous to the double-stranded target DNA.
  • the sequence in the first newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence in the second newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA.
  • the sequence in the first newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA further comprises a region that is not complementary to the sequence in the second newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA.
  • the sequence in the second newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence in the first newly synthesized single-stranded DNA that is WSGR Docket No.59761-772601 exogenous to the double-stranded target DNA.
  • the sequence in the second newly synthesized single-stranded DNA that is exogenous to the double-stranded target DNA further comprises a region that is not complementary to the sequence in the first newly synthesized single- stranded DNA that is exogenous to the double-stranded target DNA.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA form an OD that comprises a sequence that is exogenous to the double-stranded target DNA, e.g., the TRAC gene.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA form an RD that comprises a sequence that is exogenous to the double-stranded target DNA, e.g., the TRAC gene.
  • the IND is excised and is replaced by the RD.
  • the IND is excised and is replaced by the RD.
  • the IND in the target gene e.g. the TRAC gene
  • the exogenous sequence e.g., one or more recombinase recognition sequences.
  • the first editing template comprises a sequence that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the second editing template comprises a sequence that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the sequence of the first editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence of the second editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the sequence of the first editing template that complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA further comprises a region that is not complementary to the sequence of the second editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA.
  • sequence of the second editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence of the first editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the sequence of the second editing template that has complementarity or substantial complementarity to an endogenous sequence of the double-stranded target DNA further comprises a region that is not complementary to the sequence of the first editing template that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA.
  • the first newly synthesized single-stranded DNA comprises a sequence that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the second newly synthesized single-stranded DNA comprises a sequence that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the first newly synthesized single-stranded DNA and/or the second newly synthesized single-stranded DNA comprises a sequence that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA.
  • the first newly synthesized single-stranded DNA comprises a sequence that has identity or substantial identity to an endogenous sequence on the second strand of the double-stranded target DNA, e.g., the TRAC gene.
  • the second newly synthesized single-stranded DNA comprises a sequence that has identity or substantial identity to an endogenous sequence on the first strand of the double-stranded target DNA, e.g., the TRAC gene.
  • the sequence of the first newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence of the second newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the sequence of the first newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA further comprises a region that is not complementary to the sequence of the second newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA.
  • the sequence of the second newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA comprises a region of complementarity or substantial complementarity to the sequence of the first newly synthesized single- stranded DNA that has identity or substantial identity to an endogenous sequence of the double- stranded target DNA. In some embodiments, the sequence of the second newly synthesized single- stranded DNA that has identity or substantial identity to an endogenous sequence of the double- stranded target DNA further comprises a region that is not complementary to the sequence of the first newly synthesized single-stranded DNA that has identity or substantial identity to an endogenous sequence of the double-stranded target DNA.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA form an OD that comprises an endogenous sequence of the double-stranded target DNA, e.g. the TRAC gene.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA form an RD that comprises an endogenous sequence of the double-stranded target DNA, e.g. the TRAC gene.
  • WSGR Docket No.59761-772601 In some embodiments, the RD or the OD comprises an endogenous sequence of the double-stranded target DNA, e.g., the TRAC gene.
  • the RD or the OD comprises a sequence that is exogenous compared to the double-stranded target DNA, e.g., the TRAC gene.
  • the first editing template and/or the second editing template is partially complementary, substantially complementary, or identical to the sequence of the IND.
  • the first editing template comprises a region that is complementary or identical to a region of a sequence of the IND.
  • the first editing template comprises a region of complementarity to the sequence on the first PAM strand of the IND.
  • the first editing template further comprises a region of complementarity to the second editing template.
  • the first editing template is partially complementary, substantially complementary or identical to a sequence of the IND, and is also substantially complementary to the second editing template.
  • the second editing template comprises a region that is complementary or identical to a region of a sequence of the IND.
  • the second editing template comprises a region of complementarity to the sequence on the second PAM strand of the IND.
  • the second editing template further comprises a region of complementarity to the first editing template.
  • the second editing template is partially complementary, substantially complementary or identical to a sequence of the IND, and is also substantially complementary to the first editing template.
  • the first editing template and the second editing template each comprises a region of complementarity to a sequence of the IND.
  • the partially complementary region(s) in the first editing template and the second editing template can be in any position within the first editing template and the second editing template. Accordingly, in some embodiments, the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template are partially complementary to each other, at any position within the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template.
  • the first newly synthesized single-stranded DNA comprises a region of complementarity to the first strand of the IND, at or near the 3’ end of the first newly synthesized single-stranded DNA. In some embodiments, the first newly synthesized single-stranded DNA comprises a region of complementarity to the first strand of the IND, at or near the 5’ end of the first newly synthesized single-stranded DNA. In some embodiments, the first newly synthesized single-stranded DNA comprises a region of complementarity to the first strand of the IND, in the middle of the first newly synthesized single-stranded DNA.
  • the second newly synthesized single- stranded DNA comprises a region of complementarity to the second strand of the IND, at or near the 3’ end of the second newly synthesized single-stranded DNA. In some embodiments, the second newly synthesized single-stranded DNA comprises a region of complementarity to the second strand WSGR Docket No.59761-772601 of the IND, at or near the 5’ end of the second newly synthesized single-stranded DNA. In some embodiments, the second newly synthesized single-stranded DNA comprises a region of complementarity to the second strand of the IND, in the middle of the second newly synthesized single-stranded DNA.
  • the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA each comprises a region of complementarity to each other at the 3’ end.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region that is identical to a region of the sequence on the first PAM strand of the IND.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region that is identical to a region of the sequence on the second PAM strand of the IND.
  • the first newly synthesized single-stranded DNA comprises two or more sub regions, each of which is identical to a sub region of the sequence on the first PAM strand of the IND (as exemplified in FIG. 4D).
  • the sub regions on the first newly synthesized single-stranded DNA and/or the first PAM strand of the IND may or may not be consecutive.
  • the first newly synthesized single-stranded DNA may comprise 2 sub regions each identical to a sub region of the sequence on the first PAM strand of the IND, wherein the two sub regions of the sequence on the first PAM strand of the IND are separated by a region that does not have identity or substantial identity to the first newly synthesized single-stranded DNA.
  • the second newly synthesized single-stranded DNA comprises two or more sub regions, each of which is identical to a sub region of the sequence on the second PAM strand of the IND (as exemplified in FIG.4D).
  • the sub regions on the second newly synthesized single-stranded DNA and/or the second PAM strand of the IND may or may not be consecutive.
  • the second newly synthesized single-stranded DNA may comprise 2 sub regions each identical to a sub region of the sequence on the second PAM strand of the IND, wherein the two sub regions of the sequence on the second PAM strand of the IND are separated by a region that does not have identity or substantial identity to the second newly synthesized single-stranded DNA.
  • the region of the sequence on the first PAM strand of the IND and the region of the sequence on the second PAM strand of the IND are complementary to each other.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template are at least partially complementary to each other and can anneal to each other to form an OD.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template are substantially complementary or complementary to each other and can anneal to each other to form an OD.
  • the IND is excised, and the OD is incorporated in the double-stranded target DNA at the place of the IND excision.
  • the portion in the IND WSGR Docket No.59761-772601 that is not complementary or identical to the first editing template or the second editing template is deleted from the double-stranded target DNA.
  • the deletion is at the 3’ end of the IND. In some embodiments, the deletion is at the 5’ end of the IND. In some embodiments, the deletion is in the middle of the IND.
  • the first editing template of the first PEgRNA is at least partially complementary, substantially complementary, at least partially identical, or identical to a sequence of the double-stranded target DNA outside the IND. “Outside the IND” refers to sequences or positions of the double-stranded target DNA that are not in between the two nick sites generated by the first prime editor and the second prime editor.
  • the first editing template of the first PEgRNA comprises a region of identity to a sequence outside the IND on the second PAM strand (or the first strand) of the double-stranded target DNA.
  • the first editing template of the first PEgRNA comprises a region of identity to a sequence on the first strand of the double- stranded target DNA adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA, wherein the sequence is outside the IND. In some embodiments, the first editing template of the first PEgRNA comprises a region of identity to a sequence on the first strand of the double-stranded target DNA immediately adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA, wherein the sequence is outside the IND.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region of complementarity to a sequence on the first strand of the double-stranded target DNA adjacent or immediately adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region of complementarity to a sequence on the first strand of the double- stranded target DNA immediately adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA, wherein the sequence is outside the IND.
  • the first newly synthesized single-stranded DNA anneals with the sequence on the first strand of the double-stranded target DNA adjacent or immediately adjacent to the second nick site generated by the second prime editor.
  • the IND is excised and deleted from the double-stranded target DNA, e.g., the target gene.
  • the second editing template of the second PEgRNA is at least partially complementary, substantially complementary, at least partially identical, or identical to a sequence of the double-stranded target DNA outside the IND.
  • the second editing template of the second PEgRNA comprises a region of identity to a sequence outside the IND on the first PAM strand (or the second strand) of the double-stranded target DNA. In some embodiments, the second editing template of the second PEgRNA comprises a region of identity to a sequence on the second strand of the double-stranded target DNA adjacent to the first nick site generated by the first prime WSGR Docket No.59761-772601 editor complexed with the first PEgRNA, wherein the sequence is outside the IND.
  • the second editing template of the second PEgRNA comprises a region of identity to a sequence on the second strand of the double-stranded target DNA immediately adjacent to the first nick site generated by the first prime editor complexed with the first PEgRNA, wherein the sequence is outside the IND.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence on the second strand of the double-stranded target DNA adjacent or immediately adjacent to the first nick site generated by the first prime editor complexed with the first PEgRNA.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence on the second strand of the double-stranded target DNA adjacent or immediately adjacent to the first nick site generated by the first prime editor complexed with the first PEgRNA, wherein the sequence is outside the IND.
  • the second newly synthesized single-stranded DNA anneals with the sequence on the second strand of the double-stranded target DNA adjacent or immediately adjacent to the first nick site generated by the first prime editor.
  • the IND is excised and deleted from the double-stranded target DNA, e.g., the target gene.
  • the first editing template of the first PEgRNA comprises a region at least partially identical to a sequence on the first strand of the double-stranded target DNA immediately adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA, wherein the sequence is outside the IND.
  • the second editing template of the second PEgRNA comprises a region at least partially identical to a sequence on the second strand of the double-stranded target DNA immediately adjacent to the first nick site generated by the first prime editor complexed with the first PEgRNA, wherein the sequence is outside the IND.
  • the first editing template and the second editing template further comprise a region of complementarity or substantial complementarity to each other.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region of complementarity to a sequence on the first strand of the double-stranded target DNA, wherein the sequence immediately adjacent to the second nick site generated by the second prime editor complexed with the second PEgRNA and is outside the IND.
  • the second newly synthesized single- stranded DNA encoded by the second editing template comprises a region of complementarity or substantial complementarity to a sequence on the second strand of the double-stranded target DNA, wherein the sequence is immediately adjacent to the first nick site generated by the first prime editor complexed with the first PEgRNA and is outside the IND.
  • the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly WSGR Docket No.59761-772601 synthesized single-stranded DNA encoded by the second editing template further comprise a region of complementarity or substantial complementarity to each other, and can anneal to each other to form an OD.
  • the first newly synthesized single-stranded DNA encoded by the first editing template further comprises a region that is not complementary to the second newly synthesized single-stranded DNA encoded by the second editing template and does not have complementarity or identity to the double-stranded target DNA.
  • the second newly synthesized single-stranded DNA encoded by the second editing template further comprises a region that is not complementary to the first newly synthesized single-stranded DNA encoded by the first editing template and does not have complementarity or identity to the double-stranded target DNA, e.g., the target gene.
  • the RD comprises (i) the OD, (ii) the region of the first newly synthesized single-stranded DNA that is not complementary to the second newly synthesized single-stranded DNA and does not have complementarity or identity to the double-stranded target DNA, and a complementary sequence thereof, and (iii) the region of the second newly synthesized single-stranded DNA that is not complementary to the first newly synthesized single-stranded DNA and does not have complementarity or identity to the double-stranded target DNA, and a complementary sequence thereof.
  • the IND is excised from the double-stranded target DNA, e.g., the TRAC gene, and the RD is incorporated into the double-stranded target DNA.
  • the IND is excised and deleted from the target gene, and the RD is incorporated at the place of excision of the IND.
  • the IND is excised and deleted from the target gene, and the OD is incorporated at the place of excision of the IND.
  • the RD comprises a region of identity to an endogenous sequence of the double- stranded target DNA.
  • the OD comprises a region of identity to an endogenous sequence of the double-stranded target DNA.
  • the RD does not have sequence identity to an endogenous sequence of the double-stranded target DNA. In some embodiments, the RD is exogenous to the double-stranded target DNA, e.g., the target gene. In some embodiments, the RD has a biological function or encodes a polypeptide having a biological function. In some embodiments, the OD does not have sequence identity to an endogenous sequence of the double- stranded target DNA. In some embodiments, the OD is exogenous to the double-stranded target DNA, e.g., the target gene. In some embodiments, the OD has a biological function or encodes a polypeptide having a biological function.
  • the first editing template of the first PEgRNA comprises a region at least partially identical to a sequence of the double-stranded target DNA that is outside the IND and is not immediately adjacent to (also referred to as “distal to”) the second nick site on the second PAM WSGR Docket No.59761-772601 strand of the double-stranded target DNA.
  • the first editing template of the first PEgRNA comprises a region of identity to a sequence of double-stranded target DNA on the second PAM strand that is outside the IND and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides downstream of the second nick site.
  • the second editing template of the second PEgRNA comprises a region at least partially identical to a sequence of the double-stranded target DNA that is outside the IND and is not immediately adjacent to the first nick site on the first PAM strand of the double-stranded target DNA. In some embodiments, the second editing template of the second PEgRNA comprises a region of identity to a sequence of the double-stranded target DNA on the first PAM strand that is outside the IND and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides downstream of the first nick site.
  • the second editing template of the second PEgRNA comprises a region of identity to a sequence of the double-stranded target DNA that is outside the IND and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides upstream of the first nick site.
  • the first newly synthesized single-stranded DNA encoded by the first editing template comprises a region of complementarity to a sequence of the double-stranded target DNA that is outside the IND and is not immediately adjacent (i.e., distal) to the second nick site on the second PAM strand.
  • the first newly synthesized DNA encoded by the first editing template can anneal with the sequence that is outside the IND and is not immediately adjacent to the second nick site on the second PAM strand of the double-stranded target DNA.
  • the first newly synthesized DNA encoded by the first editing template comprises a region of complementarity to, and can anneal with a sequence of the double-stranded target DNA that is outside the IND and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides downstream of the second nick site.
  • the second newly synthesized single-stranded DNA encoded by the second editing template comprises a region of complementarity to a sequence of the first PAM strand of the double-stranded target DNA that is outside the IND and is not immediately adjacent to (also referred to as “distal to”) the first nick site on the first PAM strand.
  • the second newly synthesized DNA encoded by the second editing template can anneal with the sequence that is outside the IND and is not immediately adjacent to the first nick site on the first PAM strand of the double-stranded target DNA.
  • the second newly synthesized DNA encoded by the second editing template comprises a region of complementarity to, and can anneal with a sequence of the double-stranded target DNA that is outside the IND and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides upstream of the first nick site.
  • the IND is excised and deleted from the double- stranded target DNA, e.g., the target gene.
  • the endogenous sequence of the WSGR Docket No.59761-772601 double-stranded target DNA between the 3’ end of the sequence that is outside the IND and is distal to the second nick site on the second PAM strand and the 3’ end of the sequence of the double- stranded target DNA that is outside the IND and is distal to the first nick site on the first PAM strand of the double-stranded target DNA is excised and deleted from the double-stranded target DNA.
  • the first search target sequence is downstream of the second search target sequence.
  • the first editing template comprises a region that has complementarity or substantial complementarity to the second editing template, and optionally further comprises a region that does not have a complementarity to the second editing template.
  • the second editing template comprises a region that has complementarity or substantial complementarity to the first editing template, and optionally further comprises a region that does not have complementarity to the first editing template.
  • the first newly synthesized single-stranded DNA comprises a region of complementarity to the second newly synthesized single-stranded DNA, and optionally further comprises a region that does not have a complementarity to the second newly synthesized single-stranded DNA, wherein the first newly synthesized single-stranded DNA is downstream of the second newly synthesized single-stranded DNA.
  • the second newly synthesized single-stranded DNA comprises a region of complementarity to the first newly synthesized single- stranded DNA, and optionally further comprises a region that does not have a complementarity to the first newly synthesized single-stranded DNA, wherein the first newly synthesized single-stranded DNA is downstream of the second newly synthesized single-stranded DNA.
  • the sequence of the OD or the RD is incorporated in the double-stranded target DNA, and the IND sequence is duplicated in the double-stranded target DNA.
  • Prime Editor refers to the polypeptide or polypeptide components involved in prime editing.
  • a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity.
  • the polypeptide domain having DNA binding activity is a polypeptide domain having programmable DNA binding activity.
  • the prime editor further comprises a polypeptide domain having nuclease activity.
  • the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity.
  • the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease.
  • the term “nickase” refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target.
  • the prime editor comprises a polypeptide domain that is an inactive nuclease.
  • the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease.
  • the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA polymerase is a reverse transcriptase.
  • the prime editor comprises additional polypeptides or polypeptide domains involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5' endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation.
  • the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.
  • a prime editor may be engineered.
  • the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment.
  • the polypeptide components of a prime editor may be of different origins or from different organisms.
  • a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species.
  • a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species.
  • a prime editor may comprise a S.
  • polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein.
  • a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences.
  • a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA.
  • Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part.
  • a single polynucleotide, construct, or vector encodes the prime editor fusion protein.
  • multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein.
  • Prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector.
  • the term “prime editor complex” is used interchangeably with the term “prime editing complex” and refers to a complex comprising one or more prime editor components (e.g., a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity) complexed with a PEgRNA.
  • a prime editor comprises a nucleotide polymerase domain, e.g., a DNA polymerase domain.
  • the DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or may be a functional mutant, a functional variant, or a functional fragment thereof.
  • the polymerase domain is a template dependent polymerase domain.
  • the DNA polymerase may rely on a template polynucleotide strand, e.g., the editing template sequence, for new strand DNA synthesis.
  • the prime editor comprises a DNA-dependent DNA polymerase.
  • a prime editor having a DNA-dependent DNA polymerase can synthesize a new single-stranded DNA using a PEgRNA editing template that comprises a DNA sequence as a template.
  • the PEgRNA is a chimeric or hybrid PEgRNA, and comprises an extension arm comprising a DNA strand.
  • an “extension arm” is a polynucleotide portion of a PEgRNA that comprises an editing template and a primer binding site sequence (PBS).
  • PBS primer binding site sequence
  • an extension arm further comprises additional components, for example, a 3’ modifier.
  • the chimeric or hybrid PEgRNA may comprise an RNA portion (including the spacer and the gRNA core) and a DNA portion (the extension arm comprising the editing template that includes a strand of DNA).
  • the DNA polymerases can be wild-type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes.
  • the polymerases can be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like.
  • the polymerases can be thermostable, and can include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof.
  • the DNA polymerase is a bacteriophage polymerase, for example, a T4, T7, or phi29 DNA polymerase.
  • the DNA polymerase is an archaeal polymerase, for example, pol I type archaeal polymerase or a pol II type archaeal polymerase.
  • the DNA polymerase comprises a thermostable archaeal DNA polymerase.
  • the DNA polymerase comprises a eubacterial DNA polymerase, for example, Pol I, Pol II, or Pol III polymerase.
  • the DNA polymerase is a Pol I family DNA polymerase.
  • the DNA polymerase is an E.coli Pol I DNA polymerase.
  • the DNA polymerase is a Pol II family DNA polymerase.
  • the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase.
  • the DNA polymerase is a Pol IV family DNA polymerase.
  • the DNA polymerase is an E.coli Pol IV DNA polymerase.
  • the DNA polymerase comprises a eukaryotic DNA polymerase.
  • the DNA polymerase is a Pol-beta DNA polymerase, a Pol-lambda DNA polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase.
  • the WSGR Docket No.59761-772601 DNA polymerase is a Pol-alpha DNA polymerase.
  • the DNA polymerase is a POLA1 DNA polymerase.
  • the DNA polymerase is a POLA2 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-delta DNA polymerase. In some embodiments, the DNA polymerase is a POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a POLD3 DNA polymerase. In some embodiments, the DNA polymerase is a POLD4 DNA polymerase.
  • the DNA polymerase is a Pol-epsilon DNA polymerase. In some embodiments, the DNA polymerase is a POLE1 DNA polymerase. In some embodiments, the DNA polymerase is a POLE2 DNA polymerase. In some embodiments, the DNA polymerase is a POLE3 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-eta (POLH) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-iota (POLI) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-kappa (POLK) DNA polymerase.
  • POLH Pol-eta
  • POLI Pol-iota
  • POLK Pol-kappa
  • the DNA polymerase is a Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a human Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a viral DNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a B family DNA polymerases. In some embodiments, the DNA polymerase is a herpes simplex virus (HSV) UL30 DNA polymerase. In some embodiments, the DNA polymerase is a cytomegalovirus (CMV) UL54 DNA polymerase. [0348] In some embodiments, the DNA polymerase is an archaeal polymerase.
  • the DNA polymerase is a Family B/pol I type DNA polymerase.
  • the DNA polymerase is a homolog of Pfu from Pyrococcus furiosus.
  • the DNA polymerase is a pol II type DNA polymerase.
  • the DNA polymerase is a homolog of P. furiosus DP1/DP22-subunit polymerase.
  • the DNA polymerase lacks 5’ to 3’ nuclease activity. Suitable DNA polymerases (pol I or pol II) can be derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures.
  • the DNA polymerase comprises a thermostable archaeal DNA polymerase.
  • the thermostable DNA polymerase is isolated or derived from Pyrococcus species (furiosus, species GB-D, woesii, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus.
  • Polymerases may also be from eubacterial species.
  • the DNA polymerase is a Pol I family DNA polymerase.
  • the DNA polymerase is an E.coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA polymerase is a Pol III family DNA polymerase. In WSGR Docket No.59761-772601 some embodiments, the DNA polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol IV DNA polymerase.
  • the Pol I DNA polymerase is a DNA polymerase functional variant that lacks or has reduced 5' to 3' exonuclease activity.
  • Suitable thermostable pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus species and Thermotoga maritima such as Thermus aquaticus (Taq), Thermus thermophilus (Tth) and Thermotoga maritima (Tma UlTma).
  • a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT).
  • a RT or an RT domain may be a wild-type RT domain, a full-length RT domain, or may be a functional mutant, a functional variant, or a functional fragment thereof.
  • An RT or an RT domain of a prime editor may comprise a wild-type RT, or may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants.
  • An engineered RT may comprise sequences or amino acid changes different from a naturally occurring RT.
  • the engineered RT may have improved reverse transcription activity over a naturally occurring RT or RT domain.
  • the engineered RT may have improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity.
  • a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT.
  • a prime editor comprises a virus RT, for example, a retrovirus RT.
  • Non-limiting examples of virus RT include Moloney murine leukemia virus (M-MLV, MMLVRT, M-MLV RT, or MLVRT); human T-cell leukemia virus type 1 (HTLV-1) RT; bovine leukemia virus (BLV) RT; Rous Sarcoma Virus (RSV) RT; human immunodeficiency virus (HIV) RT, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV- T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus
  • the prime editor comprises a wild type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof.
  • Table 1A provides sequences of illustrative M-MLV RTs suitable for use with compositions and methods of the disclosure.
  • a prime editor comprises a wild-type M-MLV RT as set forth in SEQ ID NO: 1002.
  • a prime editor comprises a variant M-MLV RT as set forth in WSGR Docket No.59761-772601 SEQ ID NO: 1001
  • a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 1003.
  • Table 1A Table 1A.
  • M-MLV RT Sequence SEQ Sequence Amino acid sequence ID description NO: 1002 Wild-type M- TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQA MLV RT PLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNT PLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSH QWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQQ GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEA RKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKT GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDE
  • the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, WSGR Docket No.59761-772601 D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N as compared to a reference M-MLV RT.
  • the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 1001.
  • the M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 1002.
  • the prime editor comprises a M-MLV RT comprising one or more amino acid substitutions D200N, T330P, L603W, T306K, or W313F as compared to a reference M-MLV RT.
  • a prime editor comprises a M-MLV RT comprising amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to a reference M-MLV RT.
  • the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 1001. In some embodiments, the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 1002.
  • an RT variant may be a functional fragment of a reference RT that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a reference RT,.
  • the RT variant comprises a fragment of a reference RT, such that the fragment is about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the corresponding fragment of the reference RT.
  • the fragment is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical of the amino acid length of a corresponding reference RT (M-MLV reverse transcriptase).
  • a reference RT can be any one of the RTs shown in Table 1A.
  • the RT functional fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or more amino acids in length. [0360] In still other embodiments, the functional RT variant is truncated at the N-terminus or the C- terminus, or both, by a certain number of amino acids which results in a truncated variant which still retains sufficient DNA polymerase function. In some embodiments, the functional RT variant, e.g., a functional MMLV RT variant, is truncated at the C-terminus to abolish or reduce RNAse H activity and still retain DNA polymerase activity.
  • the function RT variant has a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at the N-terminal end compared to a reference RT, e.g., a wild-type RT.
  • a reference RT e.g., a wild-type RT.
  • the RT truncated variant has a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least WSGR Docket No.59761-772601 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at the C-terminal end compared to a reference RT, e.g., a wild-type RT.
  • a reference RT e.g., a wild-type RT.
  • the reference RT is a wild-type M-MLV RT.
  • the RT truncated variant has a truncation at the N-terminal and the C-terminal end compared to a reference RT, e.g., a wild-type RT.
  • the N-terminal truncation and the C-terminal truncation are of the same length.
  • the N-terminal truncation and the C-terminal truncation are of different lengths.
  • the prime editors disclosed herein may include a functional variant of a wild- type M-MLV reverse transcriptase.
  • the prime editor comprises a functional variant of a wild-type M-MLV RT, wherein the functional variant of M-MLV RT is truncated after amino acid position 502 compared to a reference M-MLV RT.
  • the functional variant of M-MLV RT further comprises a D200X, T306X, W313X, and/or T330X amino acid substitution compared to a reference M-MLV RT, wherein X is any amino acid other than the original amino acid in the reference M-MLV RT.
  • the functional variant of M-MLV RT further comprises a D200N, T306K, W313F, and/or T330P amino acid substitution compared to a reference M-MLV RT, wherein X is any amino acid other than the original amino acid in the reference M-MLV RT.
  • the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 1001.
  • the M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 1002.
  • a DNA sequence encoding a prime editor comprising this truncated RT is 522 bp smaller than a prime editor comprising a full-length M-MLV RT, and therefore makes its potentially useful for applications where delivery of the DNA sequence is challenging due to its size (e.g., adeno-associated virus and lentivirus delivery).
  • a prime editor comprises a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in in Table 1A.
  • the prime editor comprises a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of: amino acid sequences provided in Table 1A or a variant or fragment thereof.
  • the prime editor comprises a variant M-MLV RT that comprises an amino acid sequence set forth in SEQ ID NO: 1003.
  • the prime editor comprises a variant M-MLV RT that comprises an amino acid sequence set forth in SEQ ID NO: 1004.
  • a prime editing composition or a prime editing system disclosed herein comprises a polynucleotide (e.g., a DNA, a RNA, e.g., a mRNA) that encodes a M-MLV RT.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid WSGR Docket No.59761-772601 sequence set forth in Table 1A.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NO: 1001, 1002, 1003 or 1004.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of: the amino acid sequences provided in Table 1A.
  • the polynucleotide encodes a variant M-MLV RT that comprises an amino acid sequence that is set forth in SEQ ID NO: 1003. In some embodiments, the polynucleotide encodes a variant M-MLV RT that comprises an amino acid sequence that is set forth in SEQ ID NO: 1004. [0363]
  • a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a.
  • the prime editor comprises a retron RT.
  • Programmable DNA Binding Domain [0364]
  • the DNA-binding domain of a prime editor is a programmable DNA binding domain.
  • a programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA.
  • the DNA-binding domain is a polynucleotide programmable DNA-binding domain that can associate with a guide polynucleotide (e.g., a PEgRNA) that guides the DNA-binding domain to a specific DNA sequence, e.g., a search target sequence in a target gene.
  • the DNA-binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant thereof.
  • a DNA-binding domain may also comprise a zinc-finger protein domain.
  • a DNA-binding domain comprises a transcription activator-like effector domain (TALE).
  • TALE transcription activator-like effector domain
  • the DNA-binding domain comprises a DNA nuclease.
  • the DNA-binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g., a Cas protein.
  • the DNA-binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain.
  • ZFN zinc finger nuclease
  • TALEN transcription activator like effector domain nuclease
  • the DNA-binding domain comprises a nuclease activity.
  • the DNA-binding domain of a prime editor comprises an endonuclease domain having single-strand DNA cleavage activity.
  • the endonuclease domain may comprise a FokI nuclease domain.
  • the DNA-binding domain of a prime editor comprises a nuclease having full nuclease activity.
  • the DNA-binding domain of a prime WSGR Docket No.59761-772601 editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild-type endonuclease domain.
  • the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild-type endonuclease domain.
  • the DNA- binding domain of a prime editor has a nickase activity.
  • the DNA-binding domain of a prime editor comprises a Cas protein domain that is a nickase.
  • the Cas nickase comprises one or more amino acid substitutions in a nuclease domain that reduces or abolishes its double-strand nuclease activity but retains DNA binding activity.
  • the Cas nickase comprises an amino acid substitution in a HNH domain.
  • the Cas nickase comprises an amino acid substitution in a RuvC domain.
  • the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain.
  • a Cas protein may be a Class 1 or a Class 2 Cas protein.
  • a Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or a type VI Cas protein.
  • Cas proteins include Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a/Cpfl, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cm
  • a Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides.
  • a Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms.
  • a Cas protein, e.g., Cas9 can be from any suitable organism.
  • the organism is Streptococcus pyogenes (S. pyogenes).
  • the organism is Staphylococcus aureus (S. aureus).
  • the organism is Streptococcus thermophilus (S. thermophilus).
  • the organism is Staphylococcus lugdunensis.
  • Non-limiting examples of suitable organism include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis rougevillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, WSGR Docket No.59761-772601 Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothe
  • the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis (S. lugdunensis).
  • a Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rec
  • Torquens Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp.
  • a Cas protein e.g., Cas9
  • a Cas protein can be a wild-type or a modified form of a Cas protein.
  • a Cas protein e.g., Cas9
  • a Cas protein, e.g., Cas9 can comprise an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof relative to a corresponding wild-type version of the Cas protein.
  • a Cas protein can be a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild-type exemplary Cas protein.
  • a Cas protein e.g., Cas9, may comprise one or more domains.
  • Non-limiting examples of Cas domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains.
  • a Cas protein comprises a guide nucleic acid recognition and/or binding domain that can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.
  • a Cas protein e.g., Cas9
  • a Cas protein can comprise an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein.
  • a Cas protein comprises a single nuclease domain.
  • a Cpf1 may comprise a RuvC domain but lacks HNH domain.
  • a Cas protein comprises two nuclease domains, e.g., a Cas9 protein can comprise an HNH nuclease domain and a RuvC nuclease domain.
  • a prime editor comprises a Cas protein, e.g., Cas9, wherein all nuclease domains of the Cas protein are active.
  • a prime editor comprises a Cas protein having one or more inactive nuclease domains.
  • One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity.
  • a Cas protein, e.g., Cas9, comprising mutations in a nuclease domain has reduced (e.g., nickase) or abolished nuclease activity while maintaining its ability to target a nucleic acid locus at a search target sequence when complexed with a guide nucleic acid, e.g., a PEgRNA.
  • a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double- stranded DNA in the target gene, but not a double-strand break.
  • the Cas nickase can cleave the edit strand (i.e., the PAM strand) or the non-edit strand of the target gene, but may not WSGR Docket No.59761-772601 cleave both.
  • a prime editor comprises a Cas nickase comprising two nuclease domains (e.g., Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted.
  • the Cas nickase of a prime editor comprises a nuclease inactive RuvC domain and a nuclease active HNH domain.
  • the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain.
  • a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain, e.g., an amino acid substitution that reduces or abolishes nuclease activity of the RuvC domain.
  • the Cas9 nickase comprises a D10X amino acid substitution compared to a wild-type S. pyogenes Cas9, wherein X is any amino acid other than D.
  • a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain, e.g., an amino acid substitution that reduces or abolishes nuclease activity of the HNH domain.
  • the Cas9 nickase comprises a H840X amino acid substitution compared to a wild-type S. pyogenes Cas9, wherein X is any amino acid other than H.
  • a prime editor comprises a Cas protein that can bind to the target gene in a sequence-specific manner but lacks or has abolished nuclease activity and may not cleave either strand of a double-stranded DNA in a target gene.
  • Abolished activity or lacking activity can refer to an enzymatic activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g., wild-type Cas9 nuclease activity).
  • a Cas protein of a prime editor completely lacks nuclease activity.
  • a nuclease, e.g., Cas9, that lacks nuclease activity may be referred to as nuclease inactive or “nuclease dead” (abbreviated by “d”).
  • a nuclease dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide.
  • a dead Cas protein is a dead Cas9 protein.
  • a prime editor comprises a nuclease dead Cas protein wherein all of the nuclease domains (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein) are mutated to lack catalytic activity, or are deleted.
  • a Cas protein can be modified.
  • a Cas protein e.g., Cas9
  • Cas proteins can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and/or enzymatic activity.
  • Cas proteins can also be modified to change any other activity or property of the protein, such as stability.
  • one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein.
  • a Cas protein can be a fusion protein.
  • a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain.
  • a Cas protein can also be fused to a heterologous polypeptide providing increased or WSGR Docket No.59761-772601 decreased stability.
  • the fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.
  • the Cas protein of a prime editor is a Class 2 Cas protein.
  • the Cas protein is a type II Cas protein.
  • the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, a Cas9 protein homolog, mutant, variant, or a functional fragment thereof.
  • a Cas9, Cas9 protein, Cas9 polypeptide or a Cas9 nuclease refers to an RNA guided nuclease comprising one or more Cas9 nuclease domains and a Cas9 gRNA binding domain having the ability to bind a guide polynucleotide, e.g., a PEgRNA.
  • a Cas9 protein may refer to a wild-type Cas9 protein from any organism or a homolog, ortholog, or paralog from any organisms; any functional mutants or functional variants thereof; or any functional fragments or domains thereof.
  • a prime editor comprises a full-length Cas9 protein.
  • the Cas9 protein can generally comprises at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity to a wild-type reference Cas9 protein (e.g., Cas9 from S. pyogenes).
  • the Cas9 comprises an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof as compared to a wild-type reference Cas9 protein.
  • a Cas9 protein may comprise a Cas9 protein from Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), Streptococcus canis (Sc), Streptococcus thermophilus (St), Staphylococcus lugdunensis (Slu), Neisseria meningitidis (Nm), Campylobacter jejuni (Cj), Francisella novicida (Fn), or Treponema denticola (Td), or any Cas9 homolog or ortholog from an organism known in the art.
  • a Cas9 polypeptide is a SpCas9 polypeptide e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_038431314 or a fragment or variant thereof.
  • a Cas9 polypeptide is a SaCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. J7RUA5 or a fragment or variant thereof.
  • a Cas9 polypeptide is a ScCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. A0A3P5YA78 or a fragment or variant thereof.
  • a Cas9 polypeptide is a StCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_007896501.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a SluCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_230580236.1 or WP_250638315.1 or WP_242234150.1, WP_241435384.1, WP_002460848.1, KAK58371.1, or a fragment or variant thereof.
  • a Cas9 polypeptide is a NmCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_002238326.1 or WP_061704949.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a CjCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No.
  • a Cas9 polypeptide is a FnCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in Uniprot WSGR Docket No.59761-772601 Accession No. A0Q5Y3 or a fragment or variant thereof.
  • a Cas9 polypeptide is a TdCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP_147625065.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a chimera comprising domains from two or more of the organisms described herein or those known in the art.
  • a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP_003079701.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a Cas9 polypeptide generated by replacing a PAM interaction domain of a SpCas9 with that of a Streptococcus macacae Cas9 (Spy-mac Cas9).
  • Spy-mac Cas9 Exemplary Cas9 and Cas9 nickase variants are provided in Table 1B.
  • a prime editor comprises a DNA binding domain that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 1B.
  • the DNA binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differences e.g., mutations e.g., deletions, substitutions and/or insertions compared to any one of the amino acid sequences set forth in Table 1B.
  • a prime editor comprises a Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 1B.
  • a prime editor comprises a Cas9 protein is a Cas9 nickase that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the nickase sequences set forth in Table 1B.
  • a Cas9 protein comprises an amino acid sequence that is selected from the group consisting of the sequences set forth in Table 1B.
  • a prime editor comprises a Cas9 protein that comprises an amino acid sequence that lacks a N-terminus methionine relative to an amino acid sequence set forth Table 1B.
  • the prime editing compositions or prime editing systems disclosed herein comprises a polynucleotide (e.g., a DNA, or an RNA, e.g., an mRNA) that encodes a Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 1B.
  • a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), e.g., as according to NC_002737.2:854751-858857 or the protein encoded by UniProt Q99ZW2, e.g., as according to SEQ ID NO: 1005.
  • a prime editor comprises a Cas9 protein (e.g., a SpCas9) as according to any one of the sequences set forth in SEQ ID NOs: 1005-1008 or a variant thereof.
  • the Cas9 protein is a SpCas9.
  • a SpCas9 can be a wild type SpCas9, a SpCas9 variant, or a nickase SpCas9.
  • the SpCas9 lacks the N-terminus methionine relative to a corresponding SpCas9 (e.g., a wild type SpCas9, a SpCas9 variant or a nickase SpCas9).
  • a prime editor comprises a Cas9 protein having an amino acid sequence as according to SEQ ID NO: 1005, not including the N- terminus methionine.
  • a wild type SpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 1005.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SpCas9).
  • the Cas9 protein comprising one or more mutations relative to a wild type Cas9 (e.g., a wild type SpCas9) protein comprises an amino acid sequence set forth in SEQ ID NOs: 1006, 1007, or 1008.
  • Exemplary Streptococcus pyogenes Cas9 (SpCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 1B.
  • a prime editor comprises a Cas9 protein (e.g., a SluCas9) as according to any one of the SEQ ID NOs: 1009-1011 or a variant thereof.
  • a prime editor comprises a Cas9 protein from Staphylococcus lugdunensis (SluCas9) e.g., as according to any one of the SEQ ID NOs: 1009-1011 or a variant thereof.
  • the Cas9 protein is a SluCas9.
  • a SluCas9 can be a wild type SluCas9, a SluCas9 variant, or a nickase SluCas9.
  • the SluCas9 lacks the N-terminus methionine relative to a corresponding SluCas9 (e.g., a wild type SluCas9, a SluCas9 variant or a nickase SluCas9).
  • a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 1009, not including the N-terminus methionine.
  • a wild type SluCas9 comprises an amino acid sequence set forth in SEQ ID NO: 1009.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SluCas9).
  • the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 1010 or SEQ ID NO: 1011.
  • a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any of the SEQ ID NOs: 1012-1014, or a variant thereof.
  • a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as WSGR Docket No.59761-772601 according to any one of the SEQ ID NOs: 1012-1014, or a variant thereof.
  • the Cas9 protein is a SaCas9.
  • a SaCas9 can be a wild type SaCas9, a SaCas9 variant, or a nickase SaCas9.
  • the SaCas9 lacks the N-terminus methionine relative to a corresponding SaCas9 (e.g., a wild type SaCas9, a SaCas9 variant or a nickase SaCas9).
  • a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 1012, not including the N-terminus methionine.
  • a wild type SaCas9 comprises an amino acid sequence set forth in SEQ ID NO: 1012.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions relative to a corresponding wild type Cas9 protein (e.g., a wild type SaCas9).
  • the Cas9 protein comprising one or more mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 1013 or SEQ ID NO: 1014.
  • Exemplary Staphylococcus aureus Cas9 (SaCas9) amino acid sequence useful in the prime editors disclosed herein are provided Table 1B.
  • a prime editor comprises a Cas9 protein as according to any one of the sequences set forth in SEQ ID NOs: 1015-1023, 1030-1032 or a variant thereof.
  • the Cas9 protein is a Cas9 variant, for example, a SpCas9 variant (e.g., SpCas9-NG, SpCas9-NGA, SpRY, or SpG).
  • the Cas9 protein lacks the N-terminus methionine relative to a corresponding Cas9 protein (e.g., a Cas9 variant set forth in any one of SEQ ID NOs: 1015, 1016, 1018, 1019, 1021, 1022, 1030, or 1031).
  • a prime editor comprises a Cas9 protein (e.g., a Cas9 variant), having an amino acid sequence as according to any one of SEQ ID NOs: 1015, 1018, 1021, or 1030 not including the N-terminus methionine.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 1015, 1018, 1021, or 1030).
  • the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1016, 1017, 1019, 1020, 1022, 1023, 1031, or 1032.
  • a Cas9 protein is a chimeric Cas9, e.g., modified Cas9, e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., sRGN3.1, sRGN3.3.
  • sRGNs synthetic RNA-guided nucleases
  • the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa).
  • a modified sluCas9 shows increased editing efficiency and/or specificity relative to a sluCas9 that is not modified.
  • a modified Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in editing WSGR Docket No.59761-772601 efficiency compared to a Cas9 that is not modified.
  • a Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in specificity compared to a Cas9 that is not modified.
  • a Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in cleavage activity compared to a Cas9 that is not modified.
  • a Cas9 e.g., a sRGN shows ability to cleave a 5′-NNGG-3′ PAM-containing target.
  • a prime editor comprises a Cas9 protein (e.g., a chimeric Cas9), e.g., as according any one of the sequences set forth in SEQ ID NOs: 1024-1029, or a variant thereof.
  • a Cas9 protein e.g., a chimeric Cas9
  • Exemplary amino acid sequences of Cas9 protein (e.g., sRGN) useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 1024-1029.
  • a prime editor comprises a Cas9 protein, that lacks a N-terminus methionine relative to SEQ ID NO: 1024 or SEQ ID NO: 1027.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in SEQ ID NO: 1024 or SEQ ID NO: 1027).
  • the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1025, 1026, 1028, or 1029.
  • a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions.
  • a wild-type Cas9 protein comprises a RuvC domain and an HNH domain.
  • a prime editor comprises a nuclease active Cas9 protein that may cleave both strands of a double-stranded target DNA sequence.
  • the nuclease active Cas9 protein comprises a functional RuvC domain and a functional HNH domain.
  • a prime editor comprises a Cas9 nickase that can bind to a guide polynucleotide and recognize a target DNA, but can cleave only one strand of a double-stranded target DNA.
  • the Cas9 nickase comprises only one functional RuvC domain or one functional HNH domain.
  • a prime editor comprises a Cas9 that has a non- functional HNH domain and a functional RuvC domain.
  • the prime editor can cleave the edit strand (i.e., the PAM strand), but not the non-edit strand of a double-stranded target DNA sequence.
  • a prime editor comprises a Cas9 having a non-functional RuvC domain that can cleave the target strand (i.e., the non-PAM strand), but not the edit strand of a double-stranded target DNA sequence.
  • a prime editor comprises a Cas9 that has neither a functional RuvC domain nor a functional HNH domain, which may not cleave any strand of a double-stranded target DNA sequence.
  • a prime editor comprises a Cas9 having a mutation in the RuvC domain that reduces or abolishes the nuclease activity of the RuvC domain.
  • the Cas9 comprise a mutation at amino acid D10 as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 comprise a D10A mutation as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprise a mutation at amino acid D10, G12, and/or G17 as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprise a D10A mutation, a G12A mutation, and/or a G17A mutation as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • a prime editor comprises a Cas9 polypeptide having a mutation in the HNH domain that reduces or abolishes the nuclease activity of the HNH domain.
  • the Cas9 polypeptide comprise a mutation at amino acid H840 as compared to a wild- type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprise a H840A mutation as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid E762, D839, H840, N854, N856, N863, H982, H983, A984, D986, and/or a A987 as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprise a E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and/or a D986A mutation as compared to a wild-type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid residue R221, N394, and/or H840 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 1005).
  • the Cas9 polypeptide comprises a R221K, N394L, and/or H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 1005, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid residue R220, N393, and/or H839 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 1005) lacking a N- terminal methionine, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a R220K, N393K, and/or H839A mutation as compared to a wild type SpCas9 (as set forth in SEQ ID NO: 1005) lacking a N-terminal methionine, or a corresponding mutation thereof.
  • a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH domain and the RuvC domain that reduce or abolish the nuclease activity of both the HNH domain and the RuvC domain.
  • the prime editor comprises a nuclease inactive Cas9, or a nuclease dead Cas9 (dCas9).
  • the dCas9 comprises a H840X substitution and a D10X mutation compared to a wild-type SpCas9 as set WSGR Docket No.59761-772601 forth in SEQ ID NO: 1005 or corresponding mutations thereof, wherein X is any amino acid other than H for the H840X substitution and any amino acid other than D for the D10X substitution.
  • the dead Cas9 comprises a H840A and a D10A mutation as compared to a wild- type SpCas9 as set forth in SEQ ID NO: 1005, or corresponding mutations thereof.
  • the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein.
  • methionine- minus (Met (-)) Cas9 nickases include any one of the sequences set forth in SEQ ID NOs: 1007, 1008, 1011, 1014, 1017, 1020, 1023, 1026, 1029, 1032, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
  • the Cas9 proteins used herein may also include other Cas9 variants having at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% sequence identity to any reference Cas9 protein, including any wild-type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • any reference Cas9 protein including any wild-type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9, e.g., a wild-type Cas9.
  • the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of the reference Cas9, e.g., a wild-type Cas9.
  • a reference Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9.
  • a Cas9 fragment is a functional fragment that retains one or more Cas9 activities.
  • the Cas9 fragment is at least 100 amino acids in length.
  • the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
  • a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition.
  • a “protospacer adjacent motif” may be used to refer to a short DNA sequence immediately following the protospacer on the PAM strand of the target gene.
  • the PAM is recognized by the Cas nuclease in the prime editor during prime editing.
  • the PAM is required for target binding of the Cas protein.
  • the specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein.
  • a PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length.
  • the PAM can be a 5’ PAM (i.e., located upstream of the 5’ end of the protospacer). In other embodiments, the PAM can be a 3’ PAM (i.e., located downstream of the 5’ end of the protospacer).
  • the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5’- NGG-3’ PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities. Exemplary PAM sequences and corresponding Cas variants are described in Table 1C below.
  • the Cas protein comprises one or more of the amino acid substitutions as indicated compared to a wild-type Cas protein sequence, for example, the Cas9 as set forth in SEQ ID NO: 1005.
  • the PAM motifs as shown in Table 1C below are in the order of 5’ to 3’.
  • the Cas proteins of the disclosure can also be used to direct transcriptional control of target sequences, for example silencing transcription by sequence-specific binding to target sequences.
  • a Cas protein described herein may have one or mutations in a PAM recognition motif.
  • a Cas protein described herein may have altered PAM specificity.
  • nucleotides listed in Table 1C are represented by the base codes as provided in the Handbook on Industrial Property Information and Documentation, World Intellectual Property Organization (WIPO) Standard ST.26, Version 1.4.
  • an “R” in Table 1C represents the nucleotide A or G
  • a “W” in Table 1C represents A or T
  • a “V” refers to any one of nucleotides A, G, or C
  • an “N” refers to any one of nucleotides A, G, C, or T.
  • Table 1B refers to any one of nucleotides A, G, C, or T.
  • Exemplary Cas protein sequences SEQ ID Sequence Amino acid sequence NO: description 1 005 wild type MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG Streptococcus ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH Pyogenes RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA Cas9 DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN (SpCas9) PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK EIFFDQSKNG
  • PAM spCas9 wild-type NGG, NGA, NAG, NGNGA spCas9- VRVRFRR NG R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R spCas9-VQR (D1135V/R1335Q/T1337R ) NGA spCas9-EQR (D1135E/R1335Q/T1337R) NGA spCas9-VRER (D1135V/G1218R/R1335E/T1337R) NGCG spCas9-VRQR (D1135V, G1218R, R1335Q, T1337R) NGA Cas9-NG (L1111R, D1135V, G1218R, E1219F, A1322R, NGN T1337R, R1335
  • a prime editor comprises a SaCas9 polypeptide.
  • the SaCas9 polypeptide comprises one or more of mutations E782K, N968K, and R1015H as compared to a wild-type SaCas9.
  • a prime editor comprises a FnCas9 polypeptide, for example, a wild-type FnCas9 polypeptide or a FnCas9 polypeptide comprising one or more of mutations E1369R, E1449H, or R1556A as compared to the wild-type FnCas9.
  • a prime editor comprises a Sc Cas9, for example, a wild-type ScCas9 or a ScCas9 polypeptide comprises one or more of mutations I367K, G368D, I369K, H371L, T375S, T376G, and T1227K as compared to the wild-type ScCas9.
  • a prime editor comprises a St1 Cas9 polypeptide, a St3 Cas9 polypeptide, or a Slu Cas9 polypeptide.
  • a prime editor comprises a Cas polypeptide that comprises a circular permutant Cas variant.
  • a Cas9 polypeptide of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild-type Cas9 protein, or a Cas9 nickase) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA).
  • a Cas9 protein e.g., a wild-type Cas9 protein, or a Cas9 nickase
  • An exemplary circular permutant configuration may be N-terminus-[original C-terminus]- [original N-terminus]-C-terminus.
  • Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.
  • the circular permutants of a Cas protein may have the following structure: N-terminus-[original C-terminus] – [optional linker] – [original N-terminus]-C- terminus.
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 1005): WSGR Docket No.59761-772601 [0401] N-terminus-[1268-1368]-[optional linker]-[1-1267]-C-terminus; [0402] N-terminus-[1168-1368]-[optional linker]-[1-1167]-C-terminus; [0403] N-terminus-[1068-1368]-[optional linker]-[1-1067]-C-terminus; [0404] N-terminus-[968-1368]-[optional linker]-[1-967]-C-terminus; [0405] N-terminus-[868-1368]-[optional linker]-[1-867]-C-terminus; [0406] N-terminus-[768-1368]-[optional linker]-[1-767]-C-terminus; [
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 1005): [0416] N-terminus-[102-1368]-[optional linker]-[1-101]-C-terminus; [0417] N-terminus-[1028-1368]-[optional linker]-[1-1027]-C-terminus; [0418] N-terminus-[1041-1368]-[optional linker]-[1-1043]-C-terminus; [0419] N-terminus-[1249-1368]-[optional linker]-[1-1248]-C-terminus; [0420] N-terminus-[1300-1368]-[optional linker]-[1-1299]-C-terminus; or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 1005): [0422] N-terminus-[103-1368]-[optional linker]-[1-102]-C-terminus: [0423] N-terminus-[1029-1368]-[optional linker]-[1-1028]-C-terminus; [0424] N-terminus-[1042-1368]-[optional linker]-[1-1041]-C-terminus; [0425] N-terminus-[1250-1368]-[optional linker]-[1-1249]-C-terminus; [0426] N-terminus-[1301-1368]-[optional linker]-[1-1300]-C-terminus; or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
  • the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker.
  • the C-terminal fragment may correspond to the 95% or more of the C-terminal amino acids of a Cas9 (e.g., amino acids about 1300-1368 as set forth in SEQ ID NO: WSGR Docket No.59761-772601 1005 or corresponding amino acid positions thereof), or the 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the C-terminal amino acids of a Cas9 (e.g., SEQ ID NO: 1005 or a ortholog or a variant thereof).
  • a Cas9 e.g., amino acids about 1300-1368 as set forth in SEQ ID NO: WSGR Docket No.59761-772601 1005 or corresponding amino acid positions thereof
  • the N-terminal portion may correspond to 95% or more of the N-terminal amino acids of (e.g., amino acids about 1-1300 as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof), or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the N-terminal amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof.
  • the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker.
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 30%, 29%,28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof.
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • the C-terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310,300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof.
  • the C- terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 1005 or corresponding amino acid positions thereof.
  • circular permutant Cas9 variants may be a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S.
  • pyogenes Cas9 of SEQ ID NO: 1005 (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N- terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to precede the original N-terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue.
  • CP circular permutant
  • the CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain.
  • the CP site may be located (as set forth in SEQ ID NO: 1005 or corresponding amino acid WSGR Docket No.59761-772601 positions thereof) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282.
  • Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP 181 , Cas9-CP 199 , Cas9-CP 230 , Cas9-CP 270 , Cas9-CP 310 , Cas9-CP 1010 , Cas9-CP 1016 , Cas9-CP 1023 , Cas9-CP 1029 , Cas9- CP 1041 , Cas9-CP 1247 , Cas9-CP 1249 , and Cas9-CP 1282 , respectively.
  • a prime editor comprises a Cas9 functional variant that is of smaller molecular weight than a wild-type SpCas9 protein.
  • a smaller-sized Cas9 functional variant may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery.
  • a smaller-sized Cas9 functional variant is a Class 2 Type II Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type V Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type VI Cas protein. [0431] In some embodiments, a prime editor comprises a SpCas9 that is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons.
  • a prime editor comprises a Cas9 functional variant or functional fragment that is less than 1300 amino acids, less than 1290 amino acids, than less than 1280 amino acids, less than 1270 amino acids, less than 1260 amino acid, less than 1250 amino acids, less than 1240 amino acids, less than 1230 amino acids, less than 1220 amino acids, less than 1210 amino acids, less than 1200 amino acids, less than 1190 amino acids, less than 1180 amino acids, less than 1170 amino acids, less than 1160 amino acids, less than 1150 amino acids, less than 1140 amino acids, less than 1130 amino acids, less than 1120 amino acids, less than 1110 amino acids, less than 1100 amino acids, less than 1050 amino acids, less than 1000 amino acids, less than 950 amino acids, less than 900 amino acids, less than 850 amino acids, less than 800 amino acids, less than 750 amino acids, less than 700 amino acids, less than 650 amino acids, less than 600 amino acids, less than 550 amino acids, or less than 500 amino acids, but at least larger than
  • the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or WSGR Docket No.59761-772601 modified versions thereof, or WSGR Docket
  • the polypeptide domain having DNA binding activity can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a functional variant or fragment thereof.
  • a Cas9 a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d
  • Exemplary Cas proteins and nomenclature are shown in Table 2 below. Table 2.
  • Exemplary Cas proteins and nomenclature Legacy nomenclature Current nomenclature type II CRISPR-Cas enzymes Cas9 same type V CRISPR-Cas enzymes Cpf1 Cas12a CasX Cas12e C2c1 Cas12b1 Cas12b2 same C2c3 Cas12c CasY Cas12d C2c4 same C2c8 same C2c5 same C2c10 same C2c9 same type VI CRISPR-Cas enzymes C2c2 Cas13a Cas13d same C2c7 Cas13c C2c6 Cas13b [0433]
  • prime editors described herein may also comprise Cas proteins other than Cas9.
  • a prime editor as described herein may comprise a Cas12a (Cpf1) polypeptide or functional variants thereof.
  • the Cas12a polypeptide comprises a mutation that reduces or abolishes the endonuclease domain of the Cas12a polypeptide.
  • the Cas12a polypeptide is a Cas12a nickase.
  • the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12a polypeptide.
  • a prime editor comprises a Cas protein that is a Cas12b (C2c1) or a Cas12c (C2c3) polypeptide.
  • the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12b (C2c1) or Cas12c (C2c3) WSGR Docket No.59761-772601 protein.
  • the Cas protein is a Cas12b nickase or a Cas12c nickase.
  • the Cas protein is a Cas12e, a Cas12d, a Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or a Cas ⁇ polypeptide.
  • the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally- occurring Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or Cas ⁇ protein.
  • the Cas protein is a Cas12e, Cas12d, Cas13, or Cas ⁇ nickase.
  • a prime editor further comprises one or more nuclear localization sequence (NLS).
  • the NLS helps promote translocation of a protein into the cell nucleus.
  • a prime editor comprises a fusion protein, e.g., a fusion protein comprising a DNA binding domain and a DNA polymerase that comprises one or more NLSs.
  • one or more polypeptides of the prime editor are fused to or linked to one or more NLSs.
  • the prime editor comprises a DNA binding domain and a DNA polymerase domain that are provided in trans, wherein the DNA binding domain and/or the DNA polymerase domain is fused or linked to one or more NLSs.
  • a prime editor or prime editing composition comprises at least one NLS. In some embodiments, a prime editor or prime editing composition comprises at least two NLSs. In some embodiments, a prime editor or prime editing complex comprises at least three NLSs. In some embodiments, a prime editor or prime editing complex comprises more than 4, 5, 6, 7, 8, 9 or 10 NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLS, or they can be different NLSs. In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs. [0437] NLSs can be expressed as part of a prime editor or prime editing composition.
  • a NLS can be positioned almost anywhere in a protein's amino acid sequence, and generally comprises a short sequence of three or more or four or more amino acids.
  • the location of the NLS fusion can be at the N-terminus, the C-terminus, or positioned anywhere within a sequence of a prime editor or a component thereof (e.g., inserted between the DNA-binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequences of a prime editor fusion protein or a component thereof, in either N-terminus to C-terminus or C- terminus to N-terminus order).
  • a prime editor or a component thereof e.g., inserted between the DNA-binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequence
  • a prime editor is a fusion protein that comprises an NLS at the N terminus. In some embodiments, a prime editor is a fusion protein that comprises an NLS at the C terminus. In some embodiments, a prime editor is a fusion protein that comprises at least WSGR Docket No.59761-772601 one NLS at both the N terminus and the C terminus. In some embodiments, the prime editor is a fusion protein that comprises two NLSs at the N terminus and/or the C terminus. [0438] Any NLSs that are known in the art are also contemplated herein.
  • the NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS).
  • a nuclear localization signal (NLS) is predominantly basic.
  • the one or more NLSs of a prime editor are rich in lysine and arginine residues.
  • the one or more NLSs of a prime editor comprise proline residues.
  • NLS sequences suitable for use with methods and compositions of the disclosure are provided in Table 3A.
  • a NLS is a monopartite NLS.
  • a NLS is a SV40 large T antigen NLS comprising the sequence SEQ ID NO: 1159.
  • a NLS is a bipartite NLS.
  • a bipartite NLS comprises two basic domains separated by a spacer sequence comprising a variable number of amino acids.
  • a NLS is a bipartite NLS.
  • a bipartite NLS consists of two basic domains separated by a spacer sequence comprising a variable number of amino acids.
  • the spacer sequence amino acid sequence comprises Xenopus nucleoplasmin NLS SEQ ID NO: 1160, wherein X is any amino acid.
  • the NLS comprises a nucleoplasmin NLS sequence SEQ ID NO: 1161.
  • a NLS is a noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS.
  • a NLS comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence provided in Table 3A.
  • a NLS comprises an amino acid sequence selected from the group consisting of the amino acid sequences provided in Table 3A.
  • a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence provided in Table 3A.
  • a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence provided in Table 3A. Table 3A.
  • the prime editor may comprise a solubility-enhancement (SET) domain.
  • SET solubility-enhancement
  • a split intein comprises two halves of an intein protein, which may be referred to as a N-terminal half of an intein, or intein-N, and a C-terminal half of an intein, or intein- C, respectively.
  • the intein-N and the intein-C may each be fused to a protein domain (the N-terminal and the C-terminal exteins).
  • the exteins can be any protein or polypeptides, for example, any prime editor polypeptide component.
  • the intein-N and intein-C of a split intein can associate non-covalently to form an active intein and catalyze a trans- splicing reaction.
  • the trans-splicing reaction excises the two intein sequences and links the two extein sequences with a peptide bond.
  • the intein-N and the intein-C are spliced out, and a protein domain linked to the intein-N is fused to a protein domain linked to the intein-C essentially in same way as a contiguous intein does.
  • a split-intein is derived from a eukaryotic intein, a bacterial intein, or an archaeal intein.
  • the split intein so-derived will possess only the amino acid sequences essential for catalyzing trans-splicing reactions.
  • an intein-N or an intein-C further comprise one or more amino acid substitutions as compared to a wild-type intein-N or wild-type intein-C, for example, amino acid WSGR Docket No.59761-772601 substitutions that enhances the trans-splicing activity of the split intein.
  • the intein-C comprises 4 to 7 contiguous amino acid residues, wherein at least 4 amino acids of which are from the last ⁇ -strand of the intein from which it was derived.
  • the split intein is derived from a Ssp DnaE intein, e.g., Synechocytis sp. PCC6803, or any intein or split intein known in the art, or any functional variants or fragments thereof.
  • a prime editor comprises one or more epitope tags.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, thioredoxin (Trx) tags, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags.
  • His histidine
  • V5 tags FLAG tags
  • influenza hemagglutinin (HA) tags influenza hemagglutinin (HA)
  • the fusion protein comprises one or more His tags.
  • a prime editor comprises one or more polypeptide domains encoded by one or more reporter genes.
  • reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • a prime editor comprises one or more polypeptide domains that binds DNA molecules or binds other cellular molecules.
  • binding proteins or domains include, but are not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a prime editor further comprises additional components capable of interacting with, associating with, or capable of recruiting other components of the prime editor or the prime editing system.
  • a prime editor may comprise an RNA-protein recruitment polypeptide that can associate with an RNA-protein recruitment RNA aptamer.
  • an RNA-protein recruitment polypeptide can recruit, or be recruited by, a specific RNA sequence.
  • Non limiting examples of RNA- protein recruitment polypeptide and RNA aptamer pairs include a MS2 coat protein and a MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a telomerase Ku binding RNA motif, and a Sm7 protein and a telomerase Sm7 binding WSGR Docket No.59761-772601 RNA motif.
  • the prime editor comprises a DNA binding domain fused or linked to an RNA-protein recruitment polypeptide.
  • the prime editor comprises a DNA polymerase domain fused or linked to an RNA-protein recruitment polypeptide.
  • the DNA binding domain and the DNA polymerase domain fused to the RNA-protein recruitment polypeptide, or the DNA binding domain fused to the RNA-protein recruitment polypeptide and the DNA polymerase domain are co-localized by the corresponding RNA-protein recruitment RNA aptamer of the RNA-protein recruitment polypeptide.
  • the corresponding RNA-protein recruitment RNA aptamer is fused or linked to a portion of the PEgRNA.
  • an MS2 coat protein may be fused or linked to the DNA polymerase and a MS2 hairpin installed on the PEgRNA for co-localization of the DNA polymerase and the RNA-guided DNA binding domain (e.g., a Cas9 nickase).
  • a prime editor comprises a polypeptide domain, an MS2 coat protein (MCP or MS2cp), that recognizes an MS2 hairpin.
  • MCP MS2 coat protein
  • the nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is a sequence as set forth in SEQ ID NO: 1169.
  • Table 3B Exemplary MS2 hairpin and MCP sequences Description Sequence SEQ ID NO: MS2 hairpin GCCAACATGAGGATCACCCATGTCTGCAGGGCC 1169 MCP GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWIS 1170 SNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQ TVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKA MQGLLKDGNPIPSAIAANSGIY [0450] In some embodiments, the amino acid sequence of the MCP is: SEQ ID NO: 1170. [0451] In certain embodiments, components of a prime editor are directly fused to each other.
  • a linker can be any chemical group or a molecule linking two molecules or moieties, e.g., a DNA binding domain and a polymerase domain of a prime editor.
  • a linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker comprises a non-peptide moiety.
  • the linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length, for example, a polynucleotide sequence.
  • the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).
  • two or more components of a prime editor are linked to each other by a peptide linker.
  • a peptide linker is 5-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30- 35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length.
  • the peptide linker is 16 amino acids in length, 24 amino acids in length, 64 amino acids in length, or 96 amino acids in length.
  • a linker comprises 1-100 WSGR Docket No.59761-772601 amino acids.
  • Non-limiting examples of linkers are provided in Table 3C.
  • a linker comprises any one of the amino acid sequences set forth in Table 3C, or any combination thereof. [0454] Table 3C. Exemplary Peptide Linker Sequences SEQ ID NO.
  • Sequence 1171 (GGGGS)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1172 (G)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1173 (EAAAK)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1174 (GGS)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1175 (SGGS)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1176 (XP)n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid 1177 (GGS)n, wherein n is 1, 3, or 7 1178 SGSETPGTSESATPES 1179 SGGSSGGSSGSETPGTSESATPESSGGSSGGS 1180 SGGSGGSGGS 1181 SGGS 1182 SGGSSGGSSGSETPGTSESATPESAGSYPY
  • the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker.
  • the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester).
  • the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid.
  • the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid).
  • the linker WSGR Docket No.59761-772601 comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx).
  • the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane).
  • the linker comprises a polyethylene glycol moiety (PEG).
  • the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring.
  • the linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. Components of a prime editor may be connected to each other in any order.
  • the DNA binding domain and the DNA polymerase domain of a prime editor may be fused to form a fusion protein, or may be joined by a peptide or protein linker, in any order from the N terminus to the C terminus.
  • a prime editor comprises a DNA binding domain fused or linked to the C-terminal end of a DNA polymerase domain.
  • a prime editor comprises a DNA binding domain fused or linked to the N-terminal end of a DNA polymerase domain.
  • the prime editor comprises a fusion protein comprising the structure NH2-[DNA binding domain]-[DNA polymerase]-COOH; or NH2-[DNA polymerase]-[DNA binding domain]- COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
  • a prime editor comprises a fusion protein and a DNA polymerase domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA binding domain]-[RNA-protein recruitment polypeptide]-COOH.
  • a prime editor comprises a fusion protein and a DNA binding domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA polymerase domain]-[RNA-protein recruitment polypeptide]-COOH.
  • a prime editor fusion protein, a polypeptide component of a prime editor, or a polynucleotide encoding the prime editor fusion protein or polypeptide component may be split into an N-terminal half and a C-terminal half or polypeptides that encode the N-terminal half and the C terminal half, and provided to a target DNA in a cell separately.
  • a prime editor fusion protein may be split into a N-terminal and a C-terminal half for separate delivery in AAV vectors, and subsequently translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein.
  • separate halves of a protein or a fusion protein may each comprise a split-intein to facilitate colocalization and reformation of the complete protein or fusion protein by the mechanism of intein facilitated trans-splicing.
  • a prime editor comprises a N-terminal half fused to an intein-N, and a C-terminal half fused to an intein-C, or polynucleotides or vectors (e.g., AAV vectors) encoding each thereof.
  • the intein-N and the intein-C can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell.
  • an exemplary protein described herein may lack a methionine residue at the N- terminus.
  • a prime editor (e.g., a prime editor fusion protein) comprises one or more individual components shown in Tables 4A or 4B.
  • a prime editor is a prime editor fusion protein comprising all of the components shown in Table 4A or 4B.
  • a prime editor fusion protein comprises a Cas9(H840A) nickase and a wild type M-MLV RT.
  • a prime editor fusion protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT.
  • a prime editor fusion protein comprises a Cas9 (H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT or a variant M-MLV RT of SEQ ID NO: 1003.
  • a prime editor fusion protein comprises a Cas9 (R221K N394K H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT or a variant M-MLV RT of SEQ ID NO: 1003.
  • the amino acid sequence of an exemplary Prime editor fusion protein and its individual components in shown in Table 4B.
  • an exemplary prime editor protein may comprise an amino acid sequence as set forth in any of the SEQ ID NO: 1033 or SEQ ID NO: 1039.
  • a prime editing composition comprises a fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant M-MLV RT) having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[M- MLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and one or more desired PEgRNAs.
  • a DNA binding domain e.g., Cas9(H840A)
  • a reverse transcriptase e.g., a variant M-MLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[M- MLV_RT(D200N)(T330
  • the prime editing composition comprises a prime editor fusion protein that has the amino acid sequence of SEQ ID NO:1033.
  • Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 4A.
  • a prime editor or its components comprise an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a sequence in Table 4A.
  • a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- WSGR Docket No.59761-772601 [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA.
  • a DNA binding domain e.g., Cas9((R221K N394K H840A)
  • a reverse transcriptase e.g., a variant MMLV RT having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- WSGR Docket No
  • the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence of SEQ ID NO: 1039.
  • a prime editor or its components comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a sequence in Table 4B.
  • Table 4A The amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a sequence in Table 4B. Table 4A.
  • TRAC gene is available, for example, at UCSC human genome database, Gene ENSG00000277734.8, which sequence is incorporated herein by reference in its entirety.
  • the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing.
  • a PEgRNA comprises a spacer that is complementary or substantially complementary to a search target sequence on a target strand of the target gene.
  • the PEgRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor.
  • the PEgRNA comprises an editing template.
  • the PEgRNA comprises a primer binding site (PBS).
  • a PEgRNA comprises an extension arm that comprises an editing template and a primer binding site (PBS).
  • the editing template of a PEgRNA encodes one or more nucleotide edits compared to the endogenous TRAC gene sequence, e.g., the editing target sequence.
  • the one or more nucleotide edits comprise insertion, deletion, and/or substitution of one or more nucleotides.
  • the one or more nucleotide edits comprise insertion of an exogenous sequence, e.g., a recombinase recognition sequence (RSS).
  • the one or more nucleotide edits comprise insertion of one or more RSSs.
  • Dual prime editing involves two different PEgRNAs each complexed with a prime editor.
  • the prime editor is the same for each of the PEgRNA-prime editor complexes.
  • the prime editor is different for each of the PEgRNA-prime editor complexes.
  • each of the two PEgRNAs comprises a region of complementarity to a distinct search target sequence of the double-stranded target DNA, wherein the two distinct search target sequences are on the two complementary strands of the double-stranded target DNA.
  • the two PEgRNAs each can direct a prime editor to initiate the prime editing process on the two complementary strands of the double-stranded target DNA.
  • each of the two PEgRNAs comprises a spacer complementary to a separate search target sequence.
  • each of the two PEgRNAs anneals with a separate search target sequence through its spacer.
  • a first PEgRNA comprises a first spacer complementary to a first search target sequence on a first strand of a double-stranded target DNA, e.g., a double-stranded target gene.
  • the first strand of the double-stranded target DNA may be referred to as a first target strand, and the complementary strand referred to as the first PAM strand.
  • a first PEgRNA comprises a first gRNA core. In some embodiments, a first PEgRNA comprises a first editing template. In certain embodiments, a first PEgRNA comprises a first primer binding site (PBS) that is complementary to a free 3’ end formed at the first nick site.
  • a second PEgRNA comprises a second spacer complementary to a second search target sequence on a second strand of a double-stranded target DNA, e.g., a double- stranded target gene.
  • a second PEgRNA comprises a second gRNA core.
  • a second PEgRNA comprises a second editing template.
  • a second PEgRNA comprises a second primer binding site (PBS) that is complementary to a free 3’ end formed at the second nick site.
  • PBS primer binding site
  • dual prime editing results in replacement of the sequence between the first and second nick (i.e. the IND) by single stranded sequences encoded by the RTTs (the replacement duplex, RD) in the target gene, e.g., the TRAC gene.
  • incorporation of the one or more intended nucleotide edits encoded by the RTTs results in deletion of the IND from the double- stranded target DNA, e.g., the TRAC gene.
  • the target TRAC gene is a wild-type TRAC gene, and the IND is in any coding or non-coding region of the gene, the deletion or replacement of which results in abolishment or reduction in TRAC expression or function.
  • the IND is in a non-coding region of the TRAC gene. In some embodiments, the IND is in a coding region of the TRAC gene. In some embodiments, the IND is at a exon-intron junction, e.g., the exon 1-intron1 junction of the TRAC gene. In some embodiments, the IND position is designed such that after replacement of the IND by one or more recombinase recognition sequences (RSSs) encoded by the RD, potential exogenous transgene to be integrated mediated by a recombinase corresponding to the RSSs can be expressed using the endogenous promoter of the TRAC gene (although expression using an exogenous promoter, e.g.
  • RSSs recombinase recognition sequences
  • the first editing template of a first PEgRNA and the second editing template of a second PEgRNA comprise a region of complementarity to each other.
  • the first editing template encodes a first single stranded DNA
  • the second editing WSGR Docket No.59761-772601 template encodes a second single stranded DNA
  • the first and the second single stranded DNA comprise a region of complementarity to each other and are capable of forming a replacement duplex (RD).
  • the region of complementarity between the first editing template and the second editing template comprises a sequence that is exogenous to the TRAC gene.
  • the RD comprises a sequence that is exogenous to the TRAC gene.
  • the exogenous sequence comprises one or more recombinase recognition sequences.
  • the exogenous sequence may be a marker, expression tag, barcode or regulatory sequence.
  • the region of complementarity between the first editing template and the second editing template comprises a nucleotide sequence that is at least partially identical to a sequence in the double-stranded target DNA or target gene.
  • the region of complementarity between the first editing template and the second editing template comprises a nucleotide sequence that is at least partially identical to a sequence in the IND.
  • the first editing template comprises a region of identity to the second spacer.
  • the first editing template comprises a region of identity to the second spacer and also comprises a region of complementarity to the second editing template.
  • the first editing template may comprise nucleotides 1-17, 2-17, 3-17, 4-17, 5-17, 6-7, 7-17, or 8-17 of the second spacer wherein the second spacer is 20nt in length.
  • the second editing template comprises a region of identity to the first spacer.
  • the second editing template comprises a region of identity to the first spacer and also comprises a region of complementarity to the first editing template.
  • the second editing template may comprise nucleotides 1-17, 2-17, 3-17, 4-17, 5-17, 6-7, 7-17, or 8-17 of the first spacer wherein the first spacer is 20nt in length.
  • the first editing template of a first PEgRNA and the second editing template of a second PEgRNA do not comprise a region of complementarity to each other.
  • the first editing template of a first PEgRNA comprises region of identity to a sequence on the first target strand (or the first strand)
  • the second editing template comprises a region of identity to a sequence on the second target strand (or the second strand).
  • the first editing template of a first PEgRNA comprises a region of identity to a sequence on the first target strand immediately adjacent to and outside the IND.
  • the second editing template of a second PEgRNA comprises a region of identity to a sequence on the second target strand immediately adjacent to and outside the IND.
  • an editing template comprises one or more intended nucleotide edits to be incorporated in the double-stranded target DNA, e.g., the TRAC gene, by prime editing.
  • incorporation of the newly synthesized single-stranded DNA encoded by the editing template results in incorporation of one or more intended nucleotide edit in the double-stranded target DNA, e.g., the TRAC gene, compared to the endogenous sequence of the double-stranded target gene.
  • the one or more intended nucleotide edits comprises deletion, insertion, and/or substitution of one or more WSGR Docket No.59761-772601 nucleotides compared to the endogenous sequence of the double-stranded target gene, e.g., the TRAC gene.
  • the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the prime editor, for example, a reverse transcriptase domain.
  • the reverse transcriptase editing template may also be referred to herein as an RT template, or RTT.
  • the extension arm comprises a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis.
  • PBS primer binding site sequence
  • the PBS is complementary or substantially complementary to a free 3’ end on the edit strand of the target gene at a nick site generated by the prime editor.
  • the first PEgRNA comprises a first PBS that comprises a region of complementarity to the second strand of the double-stranded target DNA or target gene.
  • the second PEgRNA comprises a second PBS that comprises a region of complementarity to the first strand of the double-stranded target DNA or target gene.
  • the first PEgRNA comprises a first PBS that comprises a region of complementarity to the first spacer of the first PEgRNA.
  • the first PEgRNA comprises a first PBS that is at least partially complementary to the first spacer of the first PEgRNA.
  • the second PEgRNA comprises a second PBS that comprises a region of complementarity to the second spacer of the second PEgRNA.
  • the second PEgRNA comprises a second PBS that is at least partially complementary to the second spacer of the second PEgRNA.
  • a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide.
  • a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides.
  • a PEgRNA can include DNA in the spacer, the gRNA core, or the extension arm.
  • a PEgRNA comprises DNA in the spacer.
  • the entire spacer of a PEgRNA is a DNA sequence.
  • the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core.
  • the PEgRNA comprises DNA in the extension arm, for example, in the editing template.
  • An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase.
  • the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and/or the PBS sequences and DNA in the editing template.
  • Components of a PEgRNA may be arranged in a modular fashion.
  • the spacer, the primer binding site sequence (PBS) and the editing template can be interchangeably located in the 5’ portion of the PEgRNA, the 3’ portion of the PEgRNA, or in the middle of the gRNA core.
  • a PEgRNA comprises a PBS and an editing template sequence in 5’ to 3’ order.
  • the gRNA core of a PEgRNA of this disclosure may be located in between a spacer and an extension arm (i.e., the PBS WSGR Docket No.59761-772601 and editing template) of the PEgRNA.
  • the gRNA core of a PEgRNA may be located at the 3’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 5’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5’ end of an extension arm. In some embodiments, a PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, and an extension arm.
  • a PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, a PEgRNA comprises, from 5’ to 3’: an extension arm, a spacer, and a gRNA core. In some embodiments, the PEgRNA comprises, from 5’ to 3’: an editing template a PBS, a spacer, and a gRNA core. In some embodiments, a first PEgRNA comprises a structure: 5 ⁇ -[first spacer]-[first gRNA core]-[first editing template]-[first primer binding site sequence]-3’.
  • a first PEgRNA comprises a structure: 5 ⁇ -[first editing template]-[first primer binding site sequence]-[first spacer]-[first gRNA core]-3’.
  • a second PEgRNA comprises a structure: 5 ⁇ -[second spacer]-[second gRNA core]-[second editing template]-[second primer binding site sequence]-3’.
  • a second PEgRNA comprises a structure: 5 ⁇ -[second editing template]-[second primer binding site sequence]-[second spacer]-[second gRNA core]-3’.
  • a PEgRNA comprises a single polynucleotide molecule that comprises the spacer, the gRNA core, and the editing template. In some embodiments, a PEgRNA comprises a single polynucleotide molecule that comprises the spacer, the gRNA core, and the extension arm (i.e., a PBS and editing template). In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules.
  • a PEgRNA comprise a first polynucleotide molecule that comprises the spacer and a portion of the gRNA core, and a second polynucleotide molecule that comprises the rest of the gRNA core and the extension arm.
  • the gRNA core portion in the first polynucleotide molecule and the gRNA core portion in the second polynucleotide molecule are at least partly complementary to each other.
  • the PEgRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core referred to as a crRNA.
  • the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA.
  • the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other.
  • the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in FIG. 3.
  • a first spacer comprises a region that has substantial complementarity to a first search target sequence on a first target strand, or first strand, of a double-stranded target DNA, e.g., a TRAC gene.
  • the first spacer of a PEgRNA is identical or substantially identical to a protospacer sequence on the second strand of the target gene (except that WSGR Docket No.59761-772601 the protospacer sequence comprises thymine and the spacer may comprise uracil).
  • the first spacer is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a first search target sequence in the target gene.
  • the first spacer is substantially complementary to the first search target sequence.
  • a second spacer comprises a region that has substantial complementarity to a second search target sequence on the second target strand, or the second strand, of a double-stranded target DNA, e.g., a TRAC gene.
  • the second spacer of a PEgRNA is identical or substantially identical to a protospacer on the first strand of the target gene (except that the protospacer comprises thymine and the spacer may comprise uracil).
  • the second spacer is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a second search target sequence in the target gene.
  • the second spacer is substantially complementary to the second search target sequence.
  • the length of the spacer varies from at least 10 nucleotides to 100 nucleotides.
  • a spacer may be at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nu
  • the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.
  • the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 17 to 23 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, 20 to 30 nucleotides in length, 30 to 40 nucleotides in length, 40 to 50 nucleotides in length, 50 to 60 nucleotides in length, 60 to 70 nucleotides in length, 70 to 80 nucleotides in length, or 90 nucleotides to 100 nucleotides in length.
  • the spacer is 20 nucleotides in length. In some embodiments, the spacer is 17 to 18 nucleotides in length.
  • a PEgRNA sequence may further comprise additional nucleotides the precede a spacer beside a region of complementarity to genomic search target sequence.
  • a PEgRNA sequence (as well as the full PEgRNA sequence) may comprise an additional G at the 5’ end, for example, wherein the 5’ most nucleotide of the spacer (or the PEgRNA) is not a G.
  • the addition of the G at the 5’ end of the PEgRNA where the PEgRNA does not already begin with a G may enable transcription from a U6 promoter. Such an adaptation may be referred to as a transcription adaptation.
  • the extension arm of a first PEgRNA may be partially complementary to the spacer of the first PEgRNA.
  • the editing template (e.g., RTT) of a first PEgRNA is partially complementary to the spacer of the first PEgRNA.
  • the editing template (e.g., RTT) and the primer binding site (PBS) of the first PEgRNA are each partially complementary to the spacer of the first PEgRNA.
  • the extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT).
  • the extension arm of a second PEgRNA may be partially complementary to the spacer of the second PEgRNA.
  • the editing template (e.g., RTT) of a second PEgRNA is partially complementary to the spacer of the second PEgRNA.
  • the editing template (e.g., RTT) and the primer binding site (PBS) of the second PEgRNA are each partially complementary to the spacer of the second PEgRNA.
  • An extension arm of a PEgRNA may comprise a primer binding site sequence (also referred to as a primer binding site, PBS, or PBS sequence) that hybridizes with a free 3’ end of a single- stranded DNA in the target gene (e.g., the TRAC gene) generated by nicking with a prime editor.
  • the length of the PBS sequence may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. In some embodiments, the length of the primer binding site (PBS) varies from at least 2 nucleotides to 50 nucleotides.
  • a primer binding site may be at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.
  • the PBS is at least 4 nucleotides in length. In some embodiments, the PBS is at least 6 nucleotides in length. In some embodiments, the PBS is about 4 to 12 nucleotides, about 6 to 12 nucleotides, about 8 to 12 nucleotides, about 10 to 12 nucleotides, 4 to 14 nucleotides, about 6 to 14 nucleotides, about 8 to 14 nucleotides, about 10 to 14 nucleotides, about 12 to 14 nucleotides, 4 to 16 nucleotides, about 6 to 16 nucleotides, about 8 to 16 nucleotides, about 10 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleo
  • the PBS is about 7 to 15 nucleotides in length. In some embodiments, the PBS is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the PBS is 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. [0488]
  • the PBS of a first PEgRNA may be complementary or substantially complementary to a DNA sequence in the second strand of the target gene.
  • the PBS of a second PEgRNA may be complementary or substantially complementary to a DNA sequence in the first strand of the target gene.
  • a PBS may initiate synthesis of a new single-stranded DNA encoded by the editing template at the nick site.
  • a PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene (e.g., the TRAC gene).
  • a PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene (e.g., the TRAC gene).
  • An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing.
  • the length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA.
  • the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template or simply reverse transcriptase template (RTT).
  • the editing template (e.g., RTT) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the editing template is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the editing template is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the editing template comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides.
  • the editing template comprises about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 15 to 55, 15 to 60, 15 to 65, 15 to 70, 15 to 75, 15 to 80, 15 to 85, 15 to 90, 15 to 95, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140, 15 to 150, 25 to 30, 25 to 35, 25 to 40, 25 to 45, 25 to 50, 25 to 55, 25 to 60, 25 to 65, 25 to 70, 25 to 75, 25 to 80, 25 to 85, 25 to 90, 25 to 95, 25 to 100, 25 to 110, 25 to 120, 25 to 140, 25 to
  • the editing template comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the editing template comprises 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the editing template comprises no greater than 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the editing template comprises a sufficient number of nucleotides to form a sufficiently stable duplex with a sequence on the double-stranded target DNA. In some embodiments, the editing template comprises at least 10 polynucleotides.
  • the editing template comprises at least 15 polynucleotides. In some embodiments, the editing template comprises at least 20 polynucleotides.
  • an editing template may include sequences unrelated to the endogenous sequence. Such editing templates (RTTs) can enable insertion of a readily-identifiable sequence to permit rapid determination of successful editing, or can be used to improve editing efficiency by controlling insert length or GC content.
  • RTTs editing templates
  • an editing template may encode one or more recombinase recognition sequences (RSSs).
  • Such editing templates, or RTTs can enable insertion of the RSS(s) in the target gene, and the RSS(s) can be used as landing sites for recombinase mediated DNA insertion, deletion, inversion, or replacement.
  • prime editing insertion of a RSS allows integration of a DNA donor sequence mediated by a recombinase that recognizes the RSS (e.g., Bxb1), wherein the DNA donor sequence also comprises a RSS recognized by the recombinase (e.g., attP sequence).
  • prime editing insertion of two RSSs allow for deletion of the target gene sequence between the two RSSs or inversion of the target gene sequence between the two RSSs mediated by a recombinase that recognizes the two RSSs, depending on the orientation of the two RSSs.
  • prime editing insertion of two RSSs allow for cassette exchange of the target gene sequence between the two RSSs and a DNA donor sequence mediated by a corresponding recombinase, wherein the DNA donor sequence is flanked by two RSSs that are also recognized by the recombinase.
  • Exemplary RSS sequences that can be encoded by the PEgRNA or dual PEgRNA RTTs are provided in Table 5.
  • RSSs recognized by the same recombinase can be used for targeted insertion and other recombination events, for example, by inserting an attB sequence in a target TRAC gene via prime editing, and providing a Bxb1 recombinase and a circular DNA donor construct containing an attP sequence for integration of the DNA donor sequence in the TRAC gene at the attB site.
  • orthogonal recognition can be achieved by altering the central dinucleotide of the RSS.
  • the central dinucleotides of Bxb1 attB or attP sequences can be GT or GA, shown in bold in Table 5.
  • the central dinucleotides of a RSS can be any two nucleotides, where each nucleotide is A, T, G, or C. Additional WSGR Docket No.59761-772601 RSSs described herein and those known in the art, as well as corresponding recombinases, are also contemplated. [0497] Table 5.
  • RSS sequences and corresponding recombinases Recombinase recognition Corresponding sequence DNA sequence recombinase GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT Bxb1 attB (SEQ ID NO: 1187) Bxb1 GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGG Bxb1 attP TACAAACC (SEQ ID NO: 1188) Bxb1 GGCTTGTCGACGACGGCGGACTCCGTCGTCAGGATCAT Bxb1 attB-GA (SEQ ID NO: 1189) Bxb1 GGTTTGTCTGGTCAACCACCGCGGACTCAGTGGTGTACG Bxb1 attP-GA GTACAAACC (SEQ ID NO: 1190) Bxb1 CCGGTTTCCCTTCGCACCCGCACCGCGGCTTCGAGACCGT GACCTACATGCTCGAAGGGCGTATGCGCCACGAAGACCA Pa01 attB CCTCGGCAATCGCGG
  • the sequences in Table 6 are organized in pairs such that each paired sequences have a region of reverse complementarity to each other.
  • Each pair of editing templates in Table 6 is assigned a RTT Pair Number.
  • the first editing template can be based on either of the sequences in a given pair so long as the second editing template is based upon the other sequence in the pair.
  • the editing templates can comprise a complete sequence listed in Table 6.
  • the editing templates can also comprise any shorter 5’ fragments of the sequences in Table 6 so long as at least 10 nucleotides at the 5’ ends of the first and second editing templates have perfect reverse complementarity to each other.
  • the 5’ ends of the first and second editing templates have at least 15 nucleotides of perfect reverse complementarity to each other.
  • the 5’ ends of the first and second editing templates have at least 20 nucleotides of perfect reverse complementarity to each other.
  • the first editing template can be any one of the RTT#1 having a RTT pair number 2 or 3 wherein the second editing template is any one of the RTT#2 having a RTT pair number 2 or 3.
  • the first editing template can be any one of the RTT#2 having a RTT pair number 2 or 3 wherein the second editing template is any one of the RTT#1 having a RTT pair number 2 or 3.
  • a PEgRNA sequence or fragments thereof such as a spacer, PBS, or RTT sequence
  • the letter “T” or “thymine” indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5-methoxyuracil.
  • WSGR Docket No.59761-772601 [0500] Table 6. Exemplary editing template (RTT) sequences.
  • the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence. In some embodiments, the nucleotide edit is a deletion as compared to the target gene sequence. In some embodiments, the nucleotide edit is an insertion as compared to the target gene sequence. In some embodiments, the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two or more intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises three or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises four or more, five or more, or six or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises three single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence.
  • the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any WSGR Docket No.59761-772601 combination thereof, as compared to the target gene sequence.
  • a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution.
  • a nucleotide substitution comprises an A-to-guanine (G) substitution.
  • a nucleotide substitution comprises an A-to-cytosine (C) substitution.
  • a nucleotide substitution comprises a T-A substitution.
  • a nucleotide substitution comprises a T-G substitution.
  • a nucleotide substitution comprises a T-C substitution. In some embodiments, a nucleotide substitution comprises a G-to-A substitution. In some embodiments, a nucleotide substitution comprises a G-to-T substitution. In some embodiments, a nucleotide substitution comprises a G-to-C substitution. In some embodiments, a nucleotide substitution comprises a C-to-A substitution. In some embodiments, a nucleotide substitution comprises a C-to-T substitution. In some embodiments, a nucleotide substitution comprises a C-to-G substitution.
  • a nucleotide insertion is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length.
  • a nucleotide insertion is from 1 to 2 nucleotides, from 1 to 3 nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from 3 to 5 nucleotides, from 3 to 6 nucleotides, from 3 to 8 nucleotides, from 4 to 9 nucleotides, from 5 to 10 nucleotides, from 6 to 11 nucleotides, from 7 to 12 nucleotides, from 8 to 13 nucleotides, from 9 to 14 nucleotides, from 10 to 15 nucleotides, from 11 to 16 nucleotides, from 12 to 17 nucleotides, from 13 to 18 nucleotides, from 14 to 19 nucleotides, from 15 to 20 nucleotides in length.
  • a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises insertion of two nucleotides.
  • the editing template of a PEgRNA may comprise one or more intended nucleotide edits, compared to the TRAC gene to be edited. Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g., mutations) in the TRAC target gene may vary. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to or homologous to the protospacer sequence.
  • the nucleotide edit is in a region of the PEgRNA corresponding to a region of the TRAC gene outside of the protospacer sequence.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene results in a missense mutation, a nonsense mutation, a frame-shift mutation, a null mutation, or a mutation that generates a premature stop codon in the target TRAC gene that abolishes or decreases TRAC expression.
  • the one or more intended nucleotide edits introduce a frame shift mutation and/or generate one or more premature stop codons (e.g., at least 1, 2, 3, 4, 5, or more premature stop codons) in the target gene (e.g., TRAC gene). In some embodiments, the one or more intended nucleotide edits generates at least 2 premature stop codons in the target gene. In some embodiments, the one or more intended nucleotide edits generates at least 2, 3, 4, 5, or more WSGR Docket No.59761-772601 consecutive premature stop codons in the target gene (e.g., a TRAC gene).
  • incorporation of the one or more intended nucleotide edits introduces one or more recombinase sites in the TRAC gene. In some embodiments, incorporation of the one or more intended nucleotide edits results in deletion of the IND from the double-stranded target DNA, e.g., the TRAC gene. In some embodiments, incorporation of the one or more intended nucleotide edits results in deletion of the IND from, and insertion of an exogenous sequence encoded by the editing templates into the TRAC gene. In some embodiments, the exogenous sequence contains one or more RSSs, e.g., an attB sequence or an attP sequence.
  • the editing template comprises an adenine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises a uracil at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template does not comprise a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • a guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor.
  • the gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a Cas9 nickase of the prime editor.
  • a prime editor as described herein, for example, by association with a DNA binding domain, such as a Cas9 nickase of the prime editor.
  • a DNA binding domain such as a Cas9 nickase of the prime editor.
  • the gRNA core is capable of binding to a Cas9-based prime editor.
  • the gRNA core is capable of binding to a Cpf1-based prime editor.
  • the gRNA core is capable of binding to a Cas12b-based prime editor.
  • the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins.
  • the gRNA core of a PEgRNA may comprise one or more regions of a base paired “lower stem” adjacent to the spacer and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs.
  • the gRNA core may further WSGR Docket No.59761-772601 comprise a “nexus” distal from the spacer, followed by a hairpin structure, e.g., at the 3’ end, as exemplified in FIG. 3.
  • the gRNA core comprises modified nucleotides as compared to a wild-type gRNA core in the lower stem, upper stem, and/or the hairpin.
  • nucleotides in the lower stem, upper stem, and/or the hairpin regions may be modified, deleted, or replaced.
  • RNA nucleotides in the lower stem, upper stem, and/or the hairpin regions may be replaced with one or more DNA sequences.
  • the gRNA core comprises unmodified or wild-type RNA sequences in the nexus and/or the bulge regions.
  • the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU- AAAAC (SEQ ID NO: 1211) pairing element.
  • Exemplary gRNA cores sequences are found in Table 7, below. Table 7.
  • a PEgRNA comprises a gRNA core that comprises a modified direct repeat compared to the sequence of a naturally occurring CRISPR-Cas guide RNA scaffold, for example, a Cas9 gRNA scaffold.
  • the PEgRNA comprises a “flip and extension (F+E)” gRNA core, wherein one or more base pairs in a direct repeat is modified.
  • the PEgRNA comprises a first direct repeat (the first paring element or the lower stem), wherein a uracil is changed to an adenine (such that in the stem region, a U-A base pair is changed to an A-U base pair).
  • the PEgRNA comprises a first direct repeat wherein the fourth U-A base pair in the stem is changed to an A-U base pair. In some embodiments, the PEgRNA comprises a first direct repeat wherein one or more U-A base pair is changed to a G-C or C-G base pair. For example, in some embodiments, the PEgRNA comprises a first direct repeat comprising a modification to a GUUUU-AAAAC pairing element, wherein one or more of the U-A base pairs is changed to an A-U base pair, a G-C base pair, or a C-G base pair. In some embodiments, the PEgRNA comprises an extended first direct repeat.
  • gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein.
  • one or more nucleotides in the gRNA core is DNA.
  • a PEgRNA comprises an additional secondary structure at the 5’ end.
  • a PEgRNA comprises an additional secondary structure at the 3’ end.
  • Nucleotide sequences for exemplary secondary structure motifs for PEgRNAs are found in Table 8. Table 8. Exemplary PEgRNA secondary structure motifs. Although all the sequences provided in Table 8 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.
  • the secondary structure comprises a pseudoknot derived from a virus. In some embodiments, the secondary structure comprises a pseudoknot of a Moloney murine leukemia virus (M-MLV) genome (a mpknot). In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 612-632, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
  • M-MLV Moloney murine leukemia virus
  • the secondary structure comprises a nucleotide sequence of SEQ ID NO: 619, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
  • the secondary structure comprises a quadruplex.
  • the secondary structure comprises a G-quadruplex.
  • the secondary structure comprises a riboswitch aptamer.
  • the secondary structure comprises a riboswitch aptamer derived from a prequeosine-1 riboswitch aptamer.
  • the secondary structure comprises a modified prequeosine-1 riboswitch aptamer.
  • the secondary structure comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 633-638, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
  • the secondary structure comprises a nucleotide sequence of SEQ ID NO: 638, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
  • the secondary structure is linked to one or more other component of a PEgRNA via a linker.
  • the secondary structure is at the 3’ end of the PEgRNA and is linked to the 3’ end of a PBS via a linker.
  • the secondary structure is at the 5’ end of the PEgRNA and is linked to the 5’ end of a spacer via a linker.
  • the linker is a nucleotide linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the linker is 5 to 10 nucleotides in length.
  • the linker is 10 to 20 nucleotides in length.
  • the linker is 15 to 25 nucleotides in length.
  • the linker is 8 nucleotides in length.
  • the linker is designed to minimize base pairing between the linker and another component of the PEgRNA. In some embodiments, the linker is designed to minimize base pairing between the linker and the spacer. In some embodiments, the linker is designed to minimize base pairing between the linker and the PBS. In some embodiments, the linker is designed to minimize base pairing between the linker and the editing template. In some embodiments, the linker is designed to minimize base pairing between the linker and the sequence of the RNA secondary structure. In some embodiments, the linker is optimized to minimize base pairing between the linker and another component of the PEgRNA, in order of the following priority: spacer, PBS, editing template and then scaffold.
  • base paring probability is calculated using ViennaRNA 2.0 under standard parameters (37° C, 1 M NaCl, 0.05 M MgCl2).
  • the PEgRNA comprises a RNA secondary structure and/or a linker disclosed in Nelson et al. Engineered PEgRNAs improve prime editing efficiency. Nat Biotechnol. (2021), the entirety of which is incorporated herein by reference.
  • a PEgRNA is transcribed from a nucleotide encoding the PEgRNA, for example, a DNA plasmid encoding the PEgRNA.
  • the PEgRNA comprises WSGR Docket No.59761-772601 a self-cleaving element.
  • the self-cleaving element improves transcription and/or processing of the PEgRNA when transcribed form the nucleotide encoding the PEgRNA.
  • the PEgRNA comprises a hairpin or a RNA quadruplex.
  • the PEgRNA comprises a self-cleaving ribozyme element, for example, a hammerhead, a pistol, a hatchet, a hairpin, a VS, a twister, or a twister sister ribozyme.
  • the PEgRNA comprises a HDV ribozyme.
  • the PEgRNA comprises a hairpin recognized by Csy4.
  • the PEgRNA comprises an ENE motif. In some embodiments, the PEgRNA comprises an element for nuclear expression (ENE) from MALAT1 lnc RNA. In some embodiments, the PEgRNA comprises an ENE element from Kaposi’s sarcoma-associated herpesvirus (KSHV). In some embodiments, the PEgRNA comprises a 3’ box of a U1 snRNA. In some embodiments, the PEgRNA forms a circular RNA. [0519] In some embodiments, the PEgRNA comprises an RNA secondary structure or a motif that improves binding to the DNA-RNA duple or enhances PEgRNA activity.
  • ENE element for nuclear expression
  • KSHV Kaposi’s sarcoma-associated herpesvirus
  • the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, e.g., initiation of retroviral transcription.
  • the PEgRNA comprises a sequence of, or derived from, a primer binding site of a substrate of a reverse transcriptase, a polypurine tract (PPT), or a kissing loop.
  • the PEgRNA comprises a dimerization motif, a kissing loop, or a GNRA tetraloop – tetraloop receptor pair that results in circularization of the PEgRNA.
  • the PEgRNA comprises an RNA secondary structure of a motif that results in physical separation of the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.
  • the PEgRNA comprises a secondary structure or motif, e.g., a 5’ or 3’ extension in the spacer region that form a toehold or hairpin, wherein the secondary structure or motif competes favorably against annealing between the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.
  • Exemplary sequences for the first PEgRNA and the second PEgRNA are provided in Tables 9-25.
  • a PEgRNA may comprise one or more additional nucleotides.
  • a PEgRNA is produced by transcription from a template nucleotide, for example, a template plasmid.
  • a polynucleotide encoding the PEgRNA is appended with one or more additional nucleotides that improves PEgRNA function or expression, e.g., expression from a plasmid that encodes the PEgRNA.
  • a polynucleotide encoding a PEgRNA is appended with one or more additional nucleotides at the 5’ end or at the 3’ end.
  • the polynucleotide encoding the PEgRNA is appended with a guanine at the 5’ end, for example, if the first nucleotide at the 5’ end of the spacer is not a guanine.
  • a polynucleotide encoding the PEgRNA is appended with nucleotide sequence CACC WSGR Docket No.59761-772601 at the 5’ end.
  • the polynucleotide encoding the PEgRNA is appended with an additional nucleotide adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a Thymine.
  • the polynucleotide encoding the PEgRNA is appended with a PEgRNA comprises additional nucleotide sequence TTTTTT, TTTTTTT, TTTTT, or TTTT at the 3’ end.
  • the PEgRNA comprises the appended nucleotides from the transcription template. Accordingly, in some embodiments, the PEgRNA further comprises one or more nucleotides at the 5’ end or the 3’ end in addition to spacer, PBS, and RTT sequences. In some embodiments, the PEgRNA further comprises a guanine at the 5’ end, for example, when the first nucleotide at the 5’ end of the spacer is not a guanine.
  • the PEgRNA further comprises nucleotide sequence CACC at the 5’ end. In some embodiments, the PEgRNA further comprises an adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a thymine. In some embodiments, the PEgRNA further comprises nucleotide sequence UUUUUU, UUUUU, UUUUU, or UUUU at the 3’ end. [0522] A PEgRNA may also comprise optional modifiers, e.g., 3 ⁇ end modifier region and/or an 5 ⁇ end modifier region.
  • a PEgRNA comprises at least one nucleotide that is not part of a spacer, a gRNA core, or an extension arm.
  • the optional sequence modifiers could be positioned within or between any of the other regions shown, and not limited to being located at the 3 ⁇ and 5 ⁇ ends.
  • the PEgRNA comprises secondary RNA structure, such as, but not limited to, aptamers, hairpins, stem/loops, toeloops, and/or RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2 coat protein (MS2cp)).
  • a PEgRNA comprises a short stretch of uracil at the 5’ end or the 3’ end.
  • a PEgRNA comprising a 3’ extension arm comprises a “UUU” sequence at the 3’ end of the extension arm.
  • a PEgRNA comprises a toeloop sequence at the 3’ end.
  • the PEgRNA comprises a 3’ extension arm and a toeloop sequence at the 3’ end of the extension arm.
  • the PEgRNA comprises a 5’ extension arm and a toeloop sequence at the 5’ end of the extension arm.
  • the PEgRNA comprises a toeloop element having the sequence 5’-GAAANNNNN-3’, wherein N is any nucleobase.
  • the secondary RNA structure is positioned within the spacer. In some embodiments, the secondary structure is positioned within the extension arm. In some embodiments, the secondary structure is positioned within the gRNA core. In some embodiments, the secondary structure is positioned between the spacer and the gRNA core, between the gRNA core and the extension arm, or between the spacer and the extension arm. In some embodiments, the secondary structure is positioned between the PBS and the editing template. In some embodiments the secondary structure is positioned at the 3’ end or at the 5’ end of the PEgRNA.
  • the PEgRNA comprises a transcriptional termination signal at the 3 ⁇ end of the PEgRNA.
  • a PEgRNA comprises up to 50 nucleotides, up to 40 nucleotides, up to 30 nucleotides, up to 20 nucleotides, or up WSGR Docket No.59761-772601 to 10 nucleotides of optional sequence modifiers at either or both of the 3’ and 5’ ends of the PEgRNA.
  • the PEgRNA may comprise a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase.
  • the chemical linker may function to prevent reverse transcription of the gRNA core.
  • a PEgRNA of this disclosure may include modified nucleotides, e.g., chemically modified DNA or RNA nucleobases, and may include one or more nucleobase analogs (e.g., modifications which might add functionality, such as temperature resilience).
  • PEgRNAs as described herein may be chemically modified.
  • the phrase “chemical modifications,” as used herein, can include modifications which introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in DNA or RNA molecules).
  • the PEgRNAs provided in this disclosure may have undergone a chemical or biological modifications. Modifications may be made at any position within a PEgRNA, and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA. In some embodiments, chemical modifications can be structure guided modifications. In some embodiments, a chemical modification is at the 5’ end and/or the 3’ end of a PEgRNA. In some embodiments, a chemical modification may be within the spacer, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA. In some embodiments, a chemical modification may be within the spacer or the gRNA core of a PEgRNA.
  • a chemical modification may be within the 3’ most nucleotides of a PEgRNA. In some embodiments, a chemical modification may be within the 3’ most end of a PEgRNA. In some embodiments, a chemical modification may be within the 5’ most end of a PEgRNA. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5 or more chemically modified nucleotides at the 3’ end.
  • a PEgRNA comprises 1, 2, 3, 4, 5 or more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA comprises 1, 2, 3 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, 3 or more chemically modified nucleotides at the 5’ end. [0526] In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5’ end.
  • a PEgRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA comprises 1, 2, or 3 contiguous WSGR Docket No.59761-772601 chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end.
  • a PEgRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA comprises 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3’ end.
  • a PEgRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3, 4, 5, or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order.
  • a PEgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order.
  • a PEgRNA comprises one or more chemically modified nucleotides in the gRNA core.
  • the gRNA core of a PEgRNA may comprise one or more regions of a base paired lower stem, a base paired upper stem, where the lower stem and upper stem may be connected by a bulge comprising unpaired RNAs.
  • the gRNA core may further comprise a nexus distal from the spacer.
  • the gRNA core comprises one or more chemically modified nucleotides in the lower stem, upper stem, and/or the hairpin regions. In some embodiments, all of the nucleotides in the lower stem, upper stem, and/or the hairpin regions are chemically modified.
  • a chemical modification to a PEgRNA can comprise a 2′-O-thionocarbamate-protected nucleoside phosphoramidite, a 2′-O-methyl (M), a 2′-O-methyl 3′phosphorothioate (MS), or a 2′-O- methyl 3′thioPACE (MSP), or any combination thereof.
  • M 2′-O-thionocarbamate-protected nucleoside phosphoramidite
  • M 2′-O-methyl
  • MS 2′-O-methyl 3′phosphorothioate
  • MSP 2′-O- methyl 3′thioPACE
  • a chemically modified PEgRNA can comprise a ′-O-methyl (M) RNA, a 2′-O-methyl 3′phosphorothioate (MS) RNA, a 2′-O- methyl 3′thioPACE (MSP) RNA, a 2’-F RNA, a phosphorothioate bond modification, any other chemical modifications known in the art, or any combination thereof.
  • a chemical modification may also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA (e.g., modifications to one or both of the 3' and 5' ends of a guide RNA molecule).
  • RNA modifications can include the addition of bases to an RNA sequence, complexing the RNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).
  • an agent e.g., a protein or a complementary nucleic acid molecule
  • elements which change the structure of an RNA molecule e.g., which form secondary structures.
  • a prime editing composition for editing a TRAC gene contains one PEgRNA.
  • Exemplary PEgRNAs as well as corresponding components of the PEgRNAs are provided in Tables 9-25.
  • the PEgRNA comprises a sequence or a component thereof as provided in Tables 9-25, for example, a spacer sequence, a RTT sequence, and a PBS sequence from the same Table of Tables 9-25, or a PEgRNA sequence selected from any one of Tables 9-25.
  • the PEgRNA comprises a sequence or a component thereof as provided in Tables 9-25, and further comprises one or more contiguous nucleotides at the 5’ end of the RTT that are complementary with the edit strand of the TRAC gene.
  • the PEgRNA comprises a spacer sequence, a RTT sequence, and a PBS sequence from the same Table of Tables 9-25, at further comprises at least 4 contiguous nucleotides at the 5’ end of the RTT that are complementary with the edit strand of the TRAC gene. In some embodiments, the PEgRNA further comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more nucleotides at the 5’ end of the RTT that are complementary with the edit strand of the TRAC gene. Although all the sequences provided in Tables 9-25 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.
  • a prime editing composition having such PEgRNA may be used to introduce a Bxb1 RSS or a Pa01 sequence into the TRAC gene.
  • a prime editing composition or system is a dual prime editing composition or system.
  • a dual prime editing composition or system can comprise two PEgRNAs, each comprising a spacer, a gRNA core, and an extension arm comprising an editing template and a primer binding site (PBS).
  • An editing template can also be referred to as an RTT.
  • Exemplary dual prime editing PEgRNAs for editing the TRAC gene, as well as corresponding components of PEgRNAs, are provided in Tables 6-25.
  • PEgRNA spacers and PBSs designed to target the exon1/intron1 junction of the TRAC gene can result in insertion of a recombinase recognition sequence (RSS) encoded by the RTT(s) of the PEgRNA or the PEgRNA pair, wherein the position of insertion is downstream of the endogenous TRAC promoter.
  • RSS recombinase recognition sequence
  • the donor sequence When contacted with the recombinase and an exogenous DNA sequence comprising a donor sequence and a corresponding RSS, the donor sequence, e.g., a donor sequence encoding a CAR, can be integrated in the TRAC gene under the control of the endogenous TRAC gene promoter.
  • placing the donor sequence under the control of endogenous regulatory element, e.g., the TRAC promoter may reduce tonic signaling, delay or avoid accelerated cell differentiation and exhaustion, and increases the therapeutic potency of engineered immune cells, e.g., T cells.
  • Tables 9- 12 provide exemplary spacer sequences and PBS sequences of a 5’ PEgRNA (also referred to as a first PEgRNA in a pair of dual prime editing PEgRNAs), as well as exemplary 5’ PEgRNAs that contain a spacer and a PBS provided in the same table.
  • Tables 13-25 provide exemplary spacer sequences and PBS sequences of a 3’ PEgRNA (also referred to as a second PEgRNA in a pair of dual WSGR Docket No.59761-772601 prime editing PEgRNAs), as well as exemplary 3’ PEgRNAs that contain a spacer and a PBS provided in the same table.
  • Each of Tables 9-25 contains three columns.
  • the third column contains a description of the sequence.
  • Table 6 provides exemplary RTT sequences.
  • Table 7 provides exemplary gRNA core sequences.
  • Table 8 provides exemplary structural motif sequences. [0532] 5’ PEgRNAs and 3’ PEgRNAs exemplified in Tables 9-25 can be used in a dual prime editing composition or system with any prime editor containing a Cas9 protein capable of recognizing a NGG PAM sequence, wherein N is A, G, C, or T.
  • a dual prime editing composition or system for editing the TRAC gene can comprise (A) a 5’ PEgRNA (can also be referred to as a first PEgRNA) and a (B) 3’ PEgRNA (can also be referred to as a second PEgRNA), wherein the first PEgRNA comprises (i) a first spacer that comprises at its 3’ end a 5’ spacer sequence provided in any one of Tables 9-12; (ii) a first gRNA core capable of binding to a Cas9 protein; and (iii) a first extension arm comprising a first editing template and a first primer binding site (PBS), wherein the first PBS comprises at its 5’ end any 5’ PBS sequence provided in the same table as the first spacer; wherein the second PEgRNA comprises (i) a second spacer that comprises at its 3’ end a 3’ spacer sequence provided in any one of Tables 13-25; (ii) a second gRNA core capable of binding to a Cas9 protein; and (iii)
  • the 5’ PEgRNA spacers and the 3’ PEgRNA spacers exemplified in Tables 9-25 can be, for example, 17 to 22 nucleotides in length. Each spacer within a single table correspond to the same PAM sequence and nick site when used with a compatible Cas9 nickase.
  • the 5’ PEgRNA spacer and/or the 3’ PEgRNA spacer are 20 nucleotides in length.
  • the PBS of the 5’ PEgRNA and the 3’ PEgRNA can be, for example, 5 to 19 nucleotides in length.
  • the PBS is 8 to 17 nucleotides in length. In some embodiments, the PBS is 8 to 16 nucleotides in length.
  • a dual prime editing composition or system can comprise a 5’ PEgRNA and a 3’ PEgRNA, wherein the 5’ PEgRNA comprises a spacer selected from any one of Tables 9-12 and a PBS selected from the same table as the 5’ spacer, the 3’ PEgRNA comprises a spacer selected from Tables 13-25 and a PBS selected from the same table as a 3’ spacer, and wherein the RTT of the 5’ PEgRNA (the 5’ RTT) comprise a region of complementarity to the RTT of the 3’ PEgRNA (the 3’ RTT).
  • the 5’ PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g. a Cas9 having an inactivated HNH nuclease domain) to generate a first nick
  • the 3’ PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g. a Cas9 having an inactivated HNH nuclease domain) to generate a second nick on the TRAC gene.
  • contacting the target TRAC gene with the dual prime editing system can result in deletion of the sequence between the first nick and the second nick (the deleted region referred to as the IND) and insertion of the replacement duplex (RD) encoded by the 5’ RTT and the 3’ RTT.
  • the 5’ RTT and the 3’ RTT can be completely complementary to each other throughout their entire length, or they can be partially complementary, e.g., the region of complementarity can be at the 5’ ends of the 3’ RTT and the 5’ RTT.
  • the region of complementarity (also referred to herein as the overlapping duplex or the OD) between the 5’ RTT and the 3’ RTT can have various lengths and GC content.
  • the OD is about 15 to 38 base pairs in length. In some embodiments, the OD is 18 to 38 base pairs in length. In some embodiments, the OD is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 base pairs in length. In some embodiments, the OD is 20, 21, 22, 23, 24, 25, 26, 27 or more base pairs in length. In some embodiments, the OD has a GC content of at least about 27%. In some embodiments, the OD has a GC content of about 30% to about 85%. In some embodiments, the OD has a GC content of about 40% to about 70%.
  • the OD has a GC content of about 63% to about 70%.
  • the RD can also have various lengths and GC content. In some embodiments, when the 5’ RTT and the 3’ RTT are completely complementary to each other throughout their entire length, the OD and the RD have the same length and GC content. In some embodiments, the OD and the RD have different lengths and GC content. In some embodiments, the ratio of OD/RD is at least about 20%.
  • the ratio of OD/RD is about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more.
  • the ratio of OR/RD is about 52% or more.
  • the ratio of OR/RD is about 53% or more.
  • the ratio of OR/RD is about 55% or more.
  • the OD and the RD have the same sequence and length.
  • Exemplary RTT pairs are provided in Table 6. Each pair of RTT#1 and RTT#2 that have the same RTT pairing number can be used as the basis of the RTTs in a PEgRNA pair, with either RTT#1 or RTT#2 serving as the basis for the 5’ RTT and the other as the basis for the 3’ RTT. Each RTT#1 in Table 6 has a region of complementarity to the RTT#2 in the same RTT pair. In some WSGR Docket No.59761-772601 embodiments, each RTT#1 having the RTT pair number of 2 or 3 can be used with any RTT#2 having the RTT pair number of 2 or 3.
  • a pair of RTTs having the same length is selected.
  • a PEgRNA pair will comprise the full length of RTT# 1 and RTT#2 from a single RTT pair and will form an OD along their complete lengths.
  • at least one of the PEgRNA pair will comprise less than the full-length sequence of the RTT#1 and/or RTT#2 from a single RTT pair.
  • Such RTT pairs will form an OD only at the 5’ ends of the RTTs (corresponding to the 3’ ends of the newly synthesized strands).
  • the first editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the second editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the first editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the first editing template comprises a 5’ fragment of SEQ ID NO: 27, wherein the second editing template comprises a full length or 5’ fragment of SEQ ID NO: 107, wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of SEQ ID NO: 27, wherein the first editing template comprises a full length or 5’ fragment of SEQ ID NO: 107, and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the first editing template comprises a 5’ fragment of SEQ ID NO: 107, wherein the second editing template comprises a full length or 5’ fragment of SEQ ID NO: 27, wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of SEQ ID NO: 107, wherein the first editing template comprises a full length or 5’ fragment of SEQ ID NO: 27, and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • At least 15, 20, 25, 30, 35 or more nucleotides at the 5’ end of the first and second editing templates can have perfect reverse complementarity to each other.
  • 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or more nucleotides at the 5’ end of the first and the second editing templates have perfect complementarity to each other.
  • 20, 21, 22, 23, 24, 25, 26, 27 or more nucleotides at the 5’ end of the first and second editing templates can have perfect reverse complementarity to each other.
  • 20, 21, 22, 27 or more nucleotides at the 5’ end of the first and second editing templates can have perfect reverse complementarity to each other.
  • the RTT pairing information (RTT1 or RTT2) and corresponding recombinase of PEgRNA WSGR Docket No.59761-772601 sequences are indicated in the third column of Tables 9-25.
  • 5’ PEgRNA (Bxb1_38nt_RTT1) describes a 5’ PEgRNA sequence wherein the RTT has the sequence of Bxb1_38nt_RTT#1 as provided in Table 6.
  • the exemplary RTTs in Table 6 encode attB sequences or portions thereof that are recognized by Bxb1 recombinase or Pa01 recombinase.
  • the first RTT and the second RTT can also encode one or more of any other recombinase recognition sequences (RSSs) disclosed herein or known in the art.
  • RSSs recombinase recognition sequences
  • Exemplary RSS sequences are provided in Table 5.
  • RTTs designed to encode one or more RSS sequence can result in incorporation of the RSS sequence(s) in the target TRAC gene, which can be recognized by a corresponding recombinase.
  • the edited TRAC gene can undergo recombination with an exogenous DNA sequence that comprises a corresponding RSS sequence, e.g., attP sequence, that can be recognized by the same recombinase.
  • the exogenous DNA sequence comprises a donor sequence linked to the corresponding RSS sequence, and recombinase mediated recombination can result in integration of the donor sequence in the target TRAC gene.
  • Exemplary RSS sequence pairs e.g., attB-attP pairs, are provided in Table 5, where the orthogonal pairing relationship between the attB and the corresponding attP sequences are indicated by their identifiers in the first column.
  • the exemplary RTTs provided in Table 6 encode attB sequences or fragments thereof.
  • a PEgRNA or a dual prime editing PEgRNA pair that can comprise an RTT or an RTT pair exemplified in Table 6, or a fragment thereof, and can be used in a prime editing composition that further comprises an exogenous DNA sequence comprising a donor sequence and a corresponding attP sequence for integration of the donor sequence in TRAC.
  • PEgRNA sequences that comprise RTT or RTT pairs that encode an attP sequence (or a fragment thereof) and the spacer and PBS sequences described herein are also contemplated.
  • Such PEgRNA, or a pair of such PEgRNA can be used in a prime editing composition that further comprises an exogenous DNA sequence comprising a donor sequence and a corresponding attB sequence for integration of the donor sequence in TRAC.
  • the donor sequence for integration into the TRAC gene can encode any desired sequence, e.g., a chimeric antigen receptor (CAR), a T cell receptor (TCR), a B cell receptor, a NK cell receptor, a cytokine receptor, a chemokine receptor, a cytokine, a chemokine, a signaling protein, an antigen binding domain, an antibody, a kill switch, a reporter protein, a dominant negative mutant of a immune cell signaling protein, or a functional fragment or variant thereof.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • B cell receptor e.g., a B cell receptor, a NK cell receptor, a cytokine receptor, a chemokine receptor, a cytokine, a chemokine, a signaling protein, an antigen binding domain, an antibody, a kill switch, a reporter protein, a dominant negative mutant of a immune cell signaling protein, or a functional fragment or variant thereof.
  • prime editing-and-recombinase-mediated integration in the TRAC gene as described herein is targeted to specific positions in the TRAC gene and reduces the risk of unintended disruption or activation of non-target genes.
  • Prime editing-and-recombinase-mediated integration in the TRAC gene as described herein is unlikely to involve double strand breaks (DSBs) that are exposed to cellular repair mechanisms, and can result in improved integration efficiency and/or reduced risk associated with DSBs, e.g., genome rearrangements, chromothripsis, or unintended gene disruption or activation such as p53 activation.
  • DSBs double strand breaks
  • Recombination of the exogenous DNA sequence comprising an RSS at the prime edited TRAC gene comprising a corresponding RSS can result in integration of the donor sequence in the TRAC gene flanked by the recombination products of the RSS sequences.
  • the PEgRNA or PEgRNA pairs install an attB sequence in the TRAC gene, and the exogenous DNA sequence comprises a donor sequence and a corresponding attP sequence.
  • the TRAC gene comprises the following insert sequence from 5’ to 3’: attL-donor sequence-attR.
  • the PEgRNA or PEgRNA pairs install an attP sequence in the TRAC gene, and the exogenous DNA sequence comprises a donor sequence and a corresponding attB sequence.
  • the TRAC gene comprises the following insert sequence from 5’ to 3’: attR-donor sequence-attL.
  • the insert sequence comprises (i) 5’-attL-donor sequence-attR-3’ or (ii) 5’-attR-donor sequence-attL-3’.
  • the edited cells e.g., T cells provide herein comprise in the TRAC gene an insert sequence, wherein the insert sequence comprises 5’-attL-donor sequence-attR-3’.
  • Position of the insert sequence depends on the PEgRNA spacers designed for integration of the attB (or attP) sequence encoded by the RTTs.
  • the insert sequence in the edited cell is between the first nick site according to the first PEgRNA and the second nick site according to the second PEgRNA of a dual prime editing PEgRNA pair (i.e. the region corresponding to the IND according to the PEgRNA pair).
  • the insert sequence is immediately downstream of the first nick site.
  • the insert sequence is immediately upstream of the first nick site.
  • Nick site locations according to exemplary PEgRNA spacers provided herein in human chromosome 14 are provided in Table 45 below. Unless otherwise specified, chromosome locations and coding sequence positions (e.g. c.xxxx indicates position xxxx in the coding sequence of a certain gene) are as set forth in Genome Reference Consortium Human Build 38 (GrCH38).
  • a cell e.g., a T cell edited with the PEgRNA and PEgRNA components provided in Tables 9-25 and contacted with (i) a recombinase and (ii) a donor sequence linked to an attP sequence
  • a cell may comprise in the TRAC gene an insert sequence between the nick position of any 5’ spacer in Table 45 and the nick position of any 3’ spacer in Table 45, depending on the 5’ spacer and the 3’ spacer used in the first PEgRNA and the second PEgRNA, wherein the insert sequence comprises 5’- attL-donor sequence-attR-3’.
  • the edited cell comprises the insert sequence between human chromosome 14 positions 22547458 and 22547533.
  • the edited WSGR Docket No.59761-772601 cell comprises the insert sequence between human chromosome 14 positions 22547458 and 22547522. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547458 and 22547529. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547533. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547522. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547529. [0542] Exemplary sequences of attB-attP sequence pairs are provided in Table 5.
  • the attB sequence is GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1187).
  • the attP sequence is GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1188).
  • the attL sequence is GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1046).
  • the attR sequence is GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1047).
  • the recombinase is a Bxb1 recombinase.
  • the Bxb1 recombinase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1131.
  • the 5’ RTT comprises at its 3’ end nucleotides 8 to 17 of the 3’ spacer
  • the 3’ RTT comprises at its 3’ end nucleotides 8 to 17 of the 5’ spacer.
  • the 5’ PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g. a Cas9 having an inactivated HNH nuclease domain) to generate a first nick
  • the 3’ PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g.
  • the 5’ RTT comprises a region of complementarity to the endogenous TRAC sequence directly downstream of the second nick (i.e. the endogenous TRAC sequence directly downstream of nucleotide 3 of the search target sequence of the 3’ PEgRNA), and the 3’ RTT comprises a region of complementarity to the endogenous TRAC sequence directly downstream of the first nick (i.e. the endogenous TRAC sequence directly downstream of nucleotide 3 of the search target sequence of the 5’ PEgRNA).
  • the region of complementarity of the 5’ RTT to the endogenous TRAC sequence is at least about 15 nucleotides in length. In some embodiments, the region of complementarity of the 5’ RTT to the endogenous TRAC sequence is at least about 20 nucleotides in length. In some embodiments, the region of complementarity of the 5’ RTT to the endogenous TRAC sequence is at about 20 to 30 nucleotides in length.
  • the region of complementarity of the 5’ RTT to the endogenous TRAC sequence is at about 20, about 25, or about 30 nucleotides in length. In some embodiments, the region of complementarity of the 3’ RTT to the endogenous TRAC sequence is at least about 15 nucleotides in length. In some embodiments, the region of complementarity of the 3’ RTT to the endogenous TRAC sequence is at least about 20 nucleotides in length. In some embodiments, the region of complementarity of the 3’ RTT to the endogenous TRAC sequence is at about 20 to 30 nucleotides in length.
  • a dual prime editing composition or system can comprise a 5’ PEgRNA and a 3’ PEgRNA, wherein the 5’ PEgRNA comprises a spacer selected from any one of Tables 9-12 and a PBS selected from the same table as the 5’ spacer, the 3’ PEgRNA WSGR Docket No.59761-772601 comprises a spacer selected from Tables 13-25 and a PBS selected from the same table as a 3’ spacer, and wherein the RTT of the 5’ PEgRNA (the 5’ RTT) comprise a region of complementarity to the RTT of the 3’ PEgRNA (the 3’ RTT) and further comprises nucleotides (p-12) to (p-3) of the 3’ spacer, wherein p is the length of the 3’ spacer, and where
  • the 5’ PEgRNA and the 3’ PEgRNA exemplified in Tables 9-25 can comprise, from 5’ to 3’, the spacer, the gRNA core, the editing template (RTT), and the PBS.
  • the 3’ end of the RTT can be contiguous with the 5’ end of the PBS.
  • the PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule.
  • any PEgRNA exemplified in Tables 9-25 may comprise, or further comprise, a structural motif at the 5’ end of the PEgRNA and/or the 3’ end of the extension arm.
  • a structural motif that is capable of forming a secondary structure on its own, such as a hairpin, a pseudoknot, or other RNA structure or motif is used.
  • the 3’ motif is connected to the 3’ end of the PBS via a linker sequence.
  • Exemplary structural motifs are found in Table 8.
  • the 3’ motif comprises a sequence of 3 to 6 Uracil, e.g., the sequence of UUUU. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability.
  • PEgRNA sequences exemplified in Tables 9-25 may also be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide and/or by including 6 or 7 T nucleotides at the 3’ end of the extension arm in the expression cassette.
  • a nucleic acid template with a U6 promoter for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide and/or by including 6 or 7 T nucleotides at the 3’ end of the extension arm in the expression cassette.
  • the transcribed PEgRNA will contain a variable number of 3’ U nucleotides (e.g., 3-7 Us).
  • the modifications included in the selection of full length 5’ PEgRNAs included in Tables 9-25 are annotated in column 3 (Description) of the tables.
  • the gRNA core sequence of the 5’ PEgRNA and the gRNA core of the 3’ PEgRNA can comprise a gRNA core capable of binding to a Cas9 nuclease.
  • Exemplary gRNA core sequences are found in Table 7.
  • a PEgRNA comprises a gRNA core having SEQ ID NO: 590.
  • the PEgRNA comprises a gRNA core having SEQ ID NO: 604.
  • any 5’ PEgRNA and 3’ PEgRNA sequence provided in Tables 9-25 may be chemically synthesized and may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof.
  • the 5’ PEgRNA and/or the 3’ PEgRNA comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me WSGR Docket No.59761-772601 modification and a * indicates the presence of a phosphorothioate bond.
  • any synthetically produced PEgRNA may additionally contain a series of four 3’ U nucleotides.
  • the PEgRNAs described herein can be used in combination in a Prime Editing composition or system with one or more additional PEgRNAs that are designed to edit a different location in the same target gene or to edit a different gene in human genome.
  • the PEgRNAs described herein can be used in a multiplexed Prime Editing system with one or more additional PEgRNAs that are designed to edit a different gene, for example, a ⁇ -2 microglobulin (B2M) gene that encodes the common subunit of HLA class-I antigen.
  • B2M microglobulin
  • disruption of B2M gene can reduce or abolish expression of HLA class-I antigens on cell surface, resulting in HLA class-I- deficient cells, e.g., immune cells, that can evade attack by host cytotoxic T cells.
  • HLA class-I- deficient cells e.g., immune cells
  • multiplexed disruption of MHC class I protein expression and T cell receptor expression in an engineered cell, e.g., immune cell may reduce the allogenic reaction both of a host immune system and of the T cell receptor to host cells.
  • a prime editing system, composition, or method of the disclosure targeting a TRAC locus further comprises a PEgRNA designed to edit a B2M gene or a nucleic acid encoding the PEgRNA as described herein.
  • Table 26 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by introducing one or more premature in frame stop codons.
  • the PEgRNAs in Table 26 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T.
  • Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 1C. Although all the sequences provided in Table 26 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.
  • the B2M-editing PEgRNAs exemplified in Table 26 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 1063; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end (A) nucleotides 13-24 of sequence number 1079, (B) nucleotides 12-20 of sequence number 1085, or (C) nucleotides 7-17 of sequence number 1089, (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 1064.
  • PBS prime binding site
  • the PEgRNA spacer can be, for example, 17-22 nucleotides in length.
  • the B2M-editing PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 1060, 1061, 1062, or 1063.
  • the editing template can be referred to as a reverse transcription template (RTT).
  • RTTs exemplified in Table 26 are designed to disrupt the B2M gene by introducing one or more in frame WSGR Docket No.59761-772601 premature stop codons in the coding sequence of the B2M gene.
  • the editing template can encode an insertion of two consecutive stop codons or the complement thereof the two consecutive stop codons.
  • the insertion comprises a c.54insTAATAA edit.
  • An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 13-24, 12-24, 11-24, 10-24, 9-24, 8-24, 7-24, 6-24, 5-24, 4-24, 3-24, 2-24, or 1-24 of sequence number 1079.
  • the RTT comprises at its 3’ end the sequence corresponding to sequence number 1077, 1078, or 1079.
  • the editing template can encode one or more frame shift mutations relative to the wildtype B2M gene.
  • the frame shift mutation can be a c.51delC deletion or the complement thereof.
  • An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of sequence number 1085.
  • the RTT comprises at its 3’ end the sequence corresponding to sequence number 1080, 1081, 1082, 1083, 1084, or 1085.
  • the frame shift mutation can be a c.50insG insertion or the complement thereof.
  • An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of sequence number 1089.
  • the RTT comprises at its 3’ end the sequence corresponding to sequence number 1086, 1087, 1088, or 1089.
  • Nucleotide changes introduced by the edits encoded in the RTTs and PEgRNAs are indicated in the right column of Table 26.
  • the RTT comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4 contiguous nucleotides are located upstream of the 5’ most edit in the RTT.
  • the PBS of the B2M-editing PEgRNA can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 1064-1076.
  • the B2M-editing PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS.
  • the PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule.
  • Exemplary PEgRNAs provided in Table 26 can comprise a sequence corresponding to any one of sequence numbers 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, or 1120.
  • Any PEgRNA exemplified in Table 26 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides.
  • a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used.
  • a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence.
  • the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability.
  • the PEgRNA may alternatively or additionally comprise one WSGR Docket No.59761-772601 or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof.
  • the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • PEgRNA sequences exemplified in Table 26 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both.
  • Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series.
  • the modifications included in the selection of full length PEgRNAs included in Table 26 are annotated in column 3 of Table 26.
  • any of the B2M-editing PEgRNAs of Table 26 can be used in a Prime Editing system comprising any of the TRAC-editing PEgRNAs or TRAC-editing PEgRNA pairs provided herein.
  • the Prime Editing system can further comprise a nick guide RNA (ngRNA) targeting the B2M gene.
  • ngRNA nick guide RNA
  • a ngRNA can direct a Prime Editor to nick the non-edited strand (i.e. the non-PAM strand according to the PEgRNA), directing DNA repair to use the edited strand as a template, thereby increase editing efficiency.
  • the B2M-editing ngRNA can comprise a spacer comprising at its 3’ end a sequence corresponding to nucleotides 4-20 of any ngRNA spacer listed in Table 26 and a gRNA core capable of complexing with a Cas9 protein.
  • the sequence in the spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of sequence numbers 1121-1126.
  • the spacer of the B2M-editing ngRNA is a ngRNA spacer listed in Table 26.
  • the ngRNA is capable of directing a complexed Cas9 protein to bind the edit strand of the B2M gene; thus, a complexed Cas9 nickase containing a nuclease inactivating mutation in the HNH domain will nick the non-edit strand.
  • a PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit.
  • a B2M-editing PE3b ngRNA spacer in Table 26 annotated with the same * and number code as an RTT in Table 26 has perfect complementarity to the edit strand post-edit by a B2M-editing PEgRNA containing the RTT. Table 9.
  • Prime editing composition refers to compositions involved in the method of prime editing as described herein.
  • a prime editing composition may include a prime editor, e.g., a prime editor fusion protein, and a PEgRNA.
  • a prime editing composition may further comprise additional elements. Components of a prime editing composition may be combined to form a complex for prime editing, or may be kept separately, e.g., for administration purposes.
  • a prime editing composition comprises a first prime editing guide RNA (PEgRNA), a second PEgRNA, and a prime editor.
  • PEgRNA prime editing guide RNA
  • a prime editing composition comprises a first prime editing guide RNA (PEgRNA), a second PEgRNA, and a prime editor fusion protein complexed with the first PEgRNA and a prime editor fusion protein complexed with the second PEgRNA.
  • the prime editor fusion protein complexed with the first PEgRNA and the prime editor fusion protein complexed with the second PEgRNA are the identical prime editor fusion protein.
  • the prime editor fusion protein WSGR Docket No.59761-772601 complexed with the first PEgRNA and the prime editor fusion protein complexed with the second PEgRNA are different prime editor fusion proteins.
  • a prime editing composition comprises a first prime editing guide RNA (PEgRNA), a second PEgRNA, and a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through the first PEgRNA and/or the second PEgRNA.
  • the prime editing composition may comprise a prime editor comprising a DNA binding domain and a DNA polymerase domain linked to each other by an RNA-protein recruitment aptamer RNA sequence, which is linked to either or both of the first and second PEgRNAs.
  • the prime editing composition comprises a first PEgRNA, a second PEgRNA, and a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the prime editor for both the first PEgRNA and the second PEgRNA are the same.
  • the prime editing composition comprises a first PEgRNA, a second PEgRNA, and a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the prime editor for both the first PEgRNA and the second PEgRNA are different.
  • a prime editing composition comprises a first PEgRNA, a second PEgRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein.
  • a prime editing composition comprises a first PEgRNA, a second PEgRNA, and one or more polynucleotides, one or more polynucleotide constructs, or one or more vectors that encode a prime editor comprising a DNA binding domain and a DNA polymerase domain.
  • a prime editing composition comprises a first PEgRNA, a second PEgRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein.
  • a prime editing composition comprises multiple polynucleotides, polynucleotide constructs, or vectors, each of which encodes one or more prime editing composition components.
  • the first PEgRNA and/or the second PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas9 nickase, of the prime editor.
  • a prime editing composition comprises one or more polynucleotides that encode prime editor components and/or the first PEgRNA and/or the second PEgRNAs.
  • a prime editing composition comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain.
  • a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iii) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a fusion WSGR Docket No.59761-772601 protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iii) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iv) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iv) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N and (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C.
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C, (iii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iv) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain.
  • the DNA binding domain is a Cas protein domain, e.g., a Cas9 nickase.
  • the prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain, (iii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iv) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • the editing template of the first PEgRNA, the “first editing template”, and the editing template of the second PEgRNA, the “second editing template”, of a prime editing system may or may not have sequence complementarity to each other.
  • the first editing template has a region of complementarity or substantial complementarity to the second editing template.
  • the region of complementarity or substantial complementarity to the second editing template is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides in length.
  • the region of complementarity or substantial complementarity to the second editing template is about 5 to 10, 5 to WSGR Docket No.59761-772601 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 15 to 55, 15 to 60, 15 to 65, 15 to 70, 15 to 75, 15 to 80, 15 to 85, 15 to 90, 15 to 95, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140, 15 to 150, 25 to 30, 25 to 35, 25 to 40, 25 to 45, 25 to 50, 25 to 55, 25 to 60, 25 to 65, 25 to 70, 25 to 75, 25 to
  • the region of complementarity or substantial complementarity to the second editing template is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity to the second editing template is 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity to the second editing template is at most 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity to the second editing template is at least 10 nucleotides in length.
  • the region of complementarity or substantial complementarity to the second editing template is at least 15 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity to the second editing template is at least 20 nucleotides in length. [0565] In some embodiments, the first editing template has a region of complementarity to the second editing template and does not have a region of complementarity or substantial complementarity to the target double-stranded DNA sequence. In some embodiments, the second editing template has a region of complementarity to the first editing template and does not have a region of complementarity or substantial complementarity to the target double-stranded DNA sequence.
  • the first editing template comprises a region of complementarity or substantial complementarity to the target double-stranded DNA sequence. In some embodiments, the first editing template has a region of complementarity or substantial complementarity to the second WSGR Docket No.59761-772601 editing template and has a region of complementarity or substantial complementarity to the target double-stranded DNA sequence.
  • the second editing template comprises a region of complementarity or substantial complementarity to the target double-stranded DNA sequence. In some embodiments, the second editing template has a region of complementarity to the first editing template and has a region of complementarity or substantial complementarity to the target double-stranded DNA sequence.
  • the region of complementarity or substantial complementarity of the first editing template to the double-stranded target DNA sequence may or may not have the same length as the region of complementarity or substantial complementarity of the second editing template to the double-stranded target DNA sequence.
  • the region of complementarity or substantial complementarity of the first editing template to the target double-stranded DNA sequence is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95,
  • the region of complementarity or substantial complementarity of the first editing template to the target double-stranded DNA sequence is about 10 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity of the first editing template to the target double-stranded DNA sequence is about 15 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity of the first editing template to the target double-stranded DNA sequence is about 20 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity of the first editing template to the target double-stranded DNA sequence is about 21, 22, 23, 24, or 25 nucleotides in length.
  • the region of complementarity or substantial complementarity of the second editing template to the target double-stranded DNA sequence is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95,
  • the region of complementarity or substantial complementarity of the second editing template to the target double-stranded DNA sequence is about 10 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity of the second editing template to the target double-stranded DNA sequence is about 15 nucleotides in length. In some embodiments, the region of complementarity or WSGR Docket No.59761-772601 substantial complementarity of the second editing template to the target double-stranded DNA sequence is about 20 nucleotides in length. In some embodiments, the region of complementarity or substantial complementarity of the second editing template to the target double-stranded DNA sequence is about 21, 22, 23, 24, or 25 nucleotides in length.
  • the first editing template comprises a region that does not have complementarity or substantial complementarity (the non-complementarity region) to the second editing template. In some embodiments, the first editing template comprises a region of complementarity or substantial complementarity to the second editing template, and further comprises a non-complementarity region to the second editing template. In some embodiments, the first editing template comprises a region of complementarity or substantial complementarity to the second editing template, a non-complementarity region to the second editing template, and a region of complementarity or substantial complementarity to the target double-stranded DNA sequence.
  • the second editing template comprises a region that does not have complementarity or substantial complementarity (the non-complementarity region) to the first editing template. In some embodiments, the second editing template comprises a region of complementarity or substantial complementarity to the first editing template, and further comprises a non- complementarity region to the first editing template. In some embodiments, the second editing template comprises a region of complementarity or substantial complementarity to the first editing template, a non-complementarity region to the first editing template, and a region of complementarity or substantial complementarity to the target double-stranded DNA sequence.
  • the region of non-complementarity of the first editing template to the second editing template and the region of non-complementarity of the second editing template to the first editing template are of the same length. In some embodiments, the region of non- complementarity of the first editing template to the second editing template and the region of non- complementarity of the second editing template to the first editing template are of different lengths. In some embodiments, the first editing template and the second editing template both comprise a region of non-complementarity to each other. In some embodiments, the first editing template comprises a region of non-complementarity to the second editing template, and the second editing template does not comprise a region of non-complementarity to the first editing template.
  • the second editing template comprises a region of non-complementarity to the first editing template, and the first editing template does not comprise a region of non-complementarity to the second editing template.
  • the first editing template may be complementary or substantially complementary to the second editing template through its entire length, while the second editing template comprises a region that does not have complementarity to the first editing template, or vice versa.
  • the region of non-complementarity of the first editing template to the second editing template is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85,
  • the region of non-complementarity of the first editing template to the second editing template is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 or more nucleotides in length.
  • the region of non-complementarity of the second editing template to the first editing template is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 5 to 175, 5 to 200, 5 to 225, 5 to 250, 5 to 275, 5 to 300, 5 to 325, 5 to 350, 5 to 375, 5 to 400, 5 to 425, 5 to 450, 5 to 475, 5 to 500, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, 10 to 80, 10 to 85, 10 to 90, 10 to 95, 10 to 100, 10 to 110
  • the region of non-complementarity of the second editing template to the first editing template is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 ,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 or more nucleotides in length.
  • a prime editing system comprises one or more polynucleotides encoding one or more prime editor polypeptides, wherein activity of the prime editing system may be temporally regulated by controlling the timing in which the vectors are delivered.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA or both PEgRNAs may be delivered simultaneously.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA or both PEgRNAs may be delivered sequentially.
  • a polynucleotide encoding a component of a prime editing system may further comprise an element that is capable of modifying the intracellular half-life of the polynucleotide and/or modulating translational control.
  • the polynucleotide is a RNA, for example, an mRNA.
  • the half-life of the polynucleotide, e.g., the RNA may be increased.
  • the half-life of the polynucleotide e.g., the RNA may WSGR Docket No.59761-772601 be decreased.
  • the element may be capable of increasing the stability of the polynucleotide, e.g., the RNA.
  • the element may be capable of decreasing the stability of the polynucleotide, e.g., the RNA.
  • the element may be within the 3' UTR of the RNA.
  • the element may include a polyadenylation signal (PA).
  • PA polyadenylation signal
  • the element may include a cap, e.g., an upstream mRNA or PEgRNA end.
  • the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription.
  • the element may include at least one AU-rich element (ARE).
  • the AREs may be bound by ARE binding proteins in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment.
  • the destabilizing element may promote RNA decay, affect RNA stability, or activate translation.
  • the ARE may comprise 50 to 150 nucleotides in length.
  • the ARE may comprise at least one copy of the sequence AUUUA.
  • at least one ARE may be added to the 3' UTR of the RNA.
  • the element may be a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the element is a modified and/or truncated WPRE sequence that is capable of enhancing expression from the transcript.
  • the WPRE or equivalent may be added to the 3' UTR of the RNA.
  • the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts.
  • the polynucleotide e.g., a vector, encoding the PE or the PEgRNA may be self-destroyed via cleavage of a target sequence present on the polynucleotide, e.g., a vector. The cleavage may prevent continued transcription of a PE or a PEgRNA.
  • Polynucleotides encoding prime editing composition components can be DNA, RNA, or any combination thereof.
  • a polynucleotide encoding a prime editing composition component is an expression construct.
  • a polynucleotide encoding a prime editing composition component is a vector.
  • the vector is a DNA vector.
  • the vector is a plasmid.
  • the vector is a virus vector, e.g., a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or an adeno-associated virus vector (AAV).
  • AAV adeno-associated virus vector
  • polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3’ UTR, a 5’ UTR, or any combination thereof.
  • a polynucleotide WSGR Docket No.59761-772601 encoding a prime editing composition component is a messenger RNA (mRNA).
  • the mRNA comprises a Cap at the 5’ end and/or a poly A tail at the 3’ end.
  • a prime editing system comprises a first PEgRNA, a second PEgRNA, and a nuclease that recognizes the PAM sequence “NG.”
  • a PAM motif on the edit strand comprises an “NG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a first PEgRNA, a second PEgRNA, and a nuclease that recognizes the PAM sequence “NAG.”
  • a PAM motif on the edit strand comprises an “NAG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a first PEgRNA, a second PEgRNA, and a nuclease that recognizes the PAM sequence “NGA.”
  • a PAM motif on the edit strand comprises an “NGA” motif, wherein N is any nucleotide.
  • a prime editing system comprises a first PEgRNA, a second PEgRNA, and a nuclease that recognizes the PAM sequence “NNGG.”
  • a PAM motif on the edit strand comprises an “NNGG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a first PEgRNA, a second PEgRNA, and a nuclease that recognizes the PAM sequence “NNGRRT.”
  • a PAM motif on the edit strand comprises an “NNGRRT” motif, wherein N is any nucleotide and R is A or G.
  • Provided herein in some embodiments are example sequences for PEgRNA spacers, PBS, and editing templates for a prime editing system comprising a nuclease that recognizes the PAM sequence “NGG.”
  • a PAM motif on the edit strand comprises an “NGG” motif, wherein N is any nucleotide.
  • Prime editing composition comprising recombinases
  • the prime editing composition provided herein comprises one or more recombinases, or one or more polynucleotides encoding the recombinases, wherein the PEgRNA or PEgRNA pair of the prime editing composition encode insertion of one or more recombinase recognition sequences (RSSs) in the target gene, e.g., the TRAC gene.
  • RSSs recombinase recognition sequences
  • the recombinase is a tyrosine recombinase.
  • the recombinase is a serine recombinase.
  • Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2.
  • Examples of serine recombinases include, without limitation, Si74, No67, Kp03, Pa01, Nm60, BceINTa, BcytINTd, SscINTd, SacINTd, Hin, Gin, Tn3, I3-six, CinH, ParA, y6, Bxbl, OC31, TP901, TG1, pBT1, R4, pRV1, pFC1, MR11, A118, U153, and gp29.
  • Exemplary serine recombinase sequences and corresponding recombinases are provided in Table 5. Amino acid sequences of the recombinases are in Table 27 below (“*” indicates a stop codon): Table 27. Exemplary serine recombinase sequences WSGR Docket No.59761-772601 Recombinase Amino acid sequence MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFD RKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSAT EAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYF AQLQGREPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILT REQ
  • the DNA polynucleotide comprises (a) a donor sequence and (b) one or more RSSs recognized by the recombinase.
  • the prime editing composition comprises a recombinase that recognizes a first RSS introduced into the TRAC gene by prime editing, and further comprises a DNA polynucleotide comprising (a) a donor sequence and (b) a second RSS recognized by the same recombinase.
  • the first RSS is an attB sequence
  • the second RSS is an attP sequence.
  • the recombinase is Bxb1.
  • the RSS is an attB sequence recognized by a Bxb1 recombinase. In some embodiments, the RSS is an attP sequence recognized by a Bxb1 recombinase.
  • the recombinase is Pa01. In some embodiments, the RSS is an attB sequence recognized by a Pa01 recombinase. In some embodiments, the RSS is an attP sequence recognized by a Pa01 recombinase.
  • the DNA polynucleotide can be in any configuration that allows for recombination mediated by the recombinase and the RSSs.
  • the DNA polynucleotide can be a plasmid, a circular construct, or a minicircle.
  • the donor sequence can be any sequence for targeted integration into the TRAC gene.
  • the donor sequence comprises an expression cassette comprising a gene or an open reading frame (ORF) driven by one or more promoters.
  • the donor sequence comprises multiple expression cassettes each driven by one or more promoters.
  • the donor sequence comprises an expression cassette comprising two or more genes or ORFs driven by a promoter, wherein the two or more genes or ORFs are separated by a sequence encoding a self-cleaving peptide, e.g., a 2A peptide.
  • the donor sequence does not contain a promoter, but contains a splice acceptor sequence preceding the start codon of the transgene ORF.
  • a transgene or transgene ORF in the donor sequence encodes a polypeptide.
  • the polypeptide is involved binding, regulation, or activity of an immune cell, e.g., a T cell, and may function in combination or synergistically with disruption of the TRAC gene by prime editing.
  • the polypeptide is a cellular receptor or a portion thereof, e.g., a receptor that directs specific antigen binding.
  • the polypeptide may be a chimeric antigen receptor (CAR), a T cell receptor (TCR), a B cell receptor, a NK cell receptor, or a functional portion thereof.
  • CARs include a CD19 CAR, CD20 CAR, BAFF CAR, B7H2 CAR, GD2 CAR, MSLN CAR, HER2 CAR, EGFR CAR, CD70 CAR, BCMA CAR, or other CARs that are therapeutically relevant.
  • Exemplary TCRs include NY-ESO-1, AHNAK(S2580F), ERBB2(H473Y), HPV-E6, MAGE-A3/A6, E7, HA-1, or other TCRs that are therapeutically relevant.
  • the polypeptide is a cellular receptor or a functional portion thereof, e.g., a receptor that modulates immune cell activity.
  • the polypeptide is a cytokine receptor, e.g., IL2RA, IL2RB, IL7R, IL12R, IL18R, or IFNGR, or a functional portion thereof.
  • the polypeptide is a chemokine receptor, e.g., CCR2, CCR4, CCR5, CCR6, CXCR1, CXCR2, CXCR3, CXCR4, or a functional portion thereof.
  • the polypeptide is an immune checkpoint protein, e.g., PD1, CTLA4, CISH, TIGIT, TIM3, or LAG3.
  • the polypeptide is a cytokine, e.g., IL2, IL15, IL7, TNF, or IFNG.
  • the polypetide is a chemokine, WSGR Docket No.59761-772601 e.g., CCL2, CCL5, CCL17, CCL20, CCL22, CXCL2, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11, or CXCL12.
  • the polypeptide is a dominant-negative mutant of a protein involved in immune cell signaling, e.g. T cell signaling.
  • the polypeptide may be a dominant negative mutant of TGFBR, IL10R, IL35R, FOXP3, or STAT5.
  • the polypeptide is a transcription factor involved in immune cell signaling, e.g., T cell signaling.
  • the polypeptide may be BLIMP1, IL2RA, IL2RB, BCL6, ID2, C-MYB, IL6R, TCF1, EOMES, STAT1, STAT3, STAT4, STAT5, TBET, NFkB, GATA-3, IRF-4.
  • the polypeptide may be a signaling protein, e.g., LCK, SYK, or ZAP70.
  • the polypeptide is a reporter protein, e.g., a fluorescence protein.
  • the polypeptide is an epitope tag.
  • the polypeptide is a “suicide protein” or a “kill switch” that allows for specific elimination of cells containing the polypeptide, e.g., for specific elimination of edited cells after administration.
  • the polypeptide may be iCasp9, HSV-TK, or an epitope for antibody-mediated elimination (e.g., CD20 for rituximab-mediated elimination).
  • the recombinase (or polynucleotides encoding the recombinase) and/or the DNA polynucleotide may be provided to a cell containing the target TRAC gene simultaneously or sequentially.
  • the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after contacting the cell with a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs.
  • the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided at the same time a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs.
  • the recombinase is fused to, or connected to via a linker, e.g., a peptide linker, the prime editor, e.g., a prime editor fusion protein.
  • a linker e.g., a peptide linker
  • the prime editor or any component thereof and the recombinase can be encoded by DNA vectors (e.g. AAV vectors) or mRNAs.
  • a prime editing system or composition comprising (a) a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: (i) a spacer that is complementary to a search target sequence on a first strand of a target gene, (ii) a gRNA core capable of binding to a Cas9 nickase, and (iii) an extension arm comprising a primer binding site (PBS) that comprises a region of complementarity to a second strand of the target gene and an editing template encoding a recombinase recognition sequence (RSS) recognized by a Pa01 recombinase; (b) a prime editor comprising the Cas9 nickase capable of nicking the second strand of the target gene at a nick site and a reverse transcriptase, or one or more polynucleotides encoding the prime editor;
  • PBS primer binding site
  • RSS
  • the PBS comprises a region of complementarity to a region upstream of the nick site.
  • the editing template comprises a region of complementarity to WSGR Docket No.59761-772601 a region downstream of the nick site.
  • the Cas9 nickase comprises a nuclease inactivation mutation in a HNH domain.
  • a prime editing system or composition comprising (A) a first prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first PEgRNA, (B) a second PEgRNA or one or more polynucleotides encoding the second PEgRNA, (C) a prime editor comprising a Cas9 nickase having a nuclease inactivation mutation in a HNH domain and a reverse transcriptase, or one or more polynucleotides encoding the prime editor, and (D) a Pa01 recombinase or one or more polynucleotides encoding the Pa01 recombinase, wherein the first PEgRNA comprises: (i) a first spacer that is complementary to a first search target sequence on a first strand of a target gene, (ii) a first gRNA core capable of binding to the Cas9 nickase; and
  • the first editing template encodes the RSS.
  • the second editing template encodes the RSS.
  • the first editing template comprises a 5’ fragment of an RTT listed in RTT pair 2 or 3 of Table 6 and wherein the second editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of an RTT listed in RTT pair 2 or 3 of Table 6 and wherein the first editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the first PBS comprises a region of complementarity to a region upstream of a nick site in the second strand
  • the second PBS comprises a region of complementarity to a region upstream of a nick site in the first strand.
  • the prime editing system or composition further comprises a DNA polynucleotide that comprises (i) a second RSS recognized by the Pa01 recombinase, and (ii) a donor sequence.
  • the target gene is TRAC.
  • the prime editing method comprises contacting a target gene, e.g., a TRAC gene, with a PEgRNA and a prime editor (PE) polypeptide or a polynucleotide encoding the PE polypeptide described herein.
  • the dual prime editing method comprises contacting a target gene, e.g., a TRAC gene, with a first PEgRNA, a second PEgRNA and a PE polypeptide or a polynucleotide encoding the PE polypeptide described herein.
  • the target gene is double-stranded, and comprises two strands of DNA complementary to each other.
  • the contacting with the two PEgRNAs and the contacting with a prime editor are performed sequentially. In some embodiments, the contacting with a prime editor is performed after the contacting with the two PEgRNAs. In some embodiments, the contacting with the two PEgRNAs is performed after the contacting with a prime editor. In some embodiments, the contacting with the two PEgRNAs is performed simultaneously either prior to or after contacting with a prime editor. In some embodiments, the contacting with the two PEgRNAs is performed sequentially either prior to or after contacting with a prime editor. In some embodiments, the contacting with the two PEgRNAs, and the contacting with a prime editor are performed simultaneously.
  • the two PEgRNAs and the prime editor are associated in complexes prior to contacting a target gene.
  • contacting the target gene with the prime editing composition results in binding of the first PEgRNA to a first strand of the target gene, e.g., a TRAC gene.
  • contacting the target gene with the prime editing composition results in binding of the second PEgRNA to a second strand of the target gene, e.g., a TRAC gene.
  • contacting the target gene with the prime editing composition results in binding of the first PEgRNA to a first strand of the target gene and binding of the second PEgRNA to a second strand of the target gene, e.g., a TRAC gene.
  • contacting the target gene with the prime editing composition results in binding of the first PEgRNA to a first search target sequence on the first strand of the target gene upon contacting with the first PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of the second PEgRNA to a second search WSGR Docket No.59761-772601 target sequence on the second strand of the target gene upon contacting with the second PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of the first PEgRNA to a first search target sequence on the first strand of the target gene upon contacting with the first PEgRNA and binding of the second PEgRNA to a second search target sequence on the second strand of the target gene upon contacting with the second PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of a first spacer of the first PEgRNA to a first search target sequence on the first strand of the target gene upon said contacting of the first PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of a second spacer of the second PEgRNA to a second search target sequence on the second strand of the target gene upon said contacting of the second PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of a first spacer of the first PEgRNA to a first search target sequence on the first strand of the target gene upon said contacting of the first PEgRNA and binding of a second spacer of the second PEgRNA to a second search target sequence on the second strand of the target gene upon said contacting of the second PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, e.g., the target TRAC gene, upon the contacting of the PE composition with the target gene.
  • a DNA binding domain of a prime editor associates with either a first PEgRNA and/or a second PEgRNA.
  • a prime editor associated with a first PEgRNA binds the first strand of a target gene, e.g., a TRAC gene, as directed by the first PEgRNA.
  • a prime editor associated with a second PEgRNA binds the second strand of a target gene, e.g., a TRAC gene, as directed by the second PEgRNA.
  • a prime editor associated with a first PEgRNA binds the first strand of a target gene as directed by the first PEgRNA
  • a prime editor associated with a second PEgRNA binds the second strand of the target gene as directed by the second PEgRNA.
  • a first PEgRNA directs a prime editor to generate a nick on the second strand of a target gene.
  • a second PEgRNA directs a prime editor to generate a nick on the first strand of a target gene.
  • a first PEgRNA directs a prime editor to generate a first nick on the second strand of a target gene
  • a second PEgRNA directs a prime editor to generate a second nick on the first strand of a target gene, thereby generating an inter-nick duplex (IND) between the position of the first nick and the position of the second nick on the target gene.
  • the DNA binding domain of the prime editor is a Cas domain. In some embodiments, the DNA binding domain of the prime editor is a Cas9.
  • the DNA binding domain of the prime editor is a Cas9 nickase.
  • contacting the target gene with the prime editing composition results in hybridization of the PEgRNA (e.g., the first PEgRNA and/or the second PEgRNA) with the 3’ end WSGR Docket No.59761-772601 of the nicked single-stranded DNA, thereby priming DNA polymerization by a DNA polymerase domain of the prime editor.
  • the free 3’ end of the single-stranded DNA generated at the nick site hybridizes to a primer binding site sequence (PBS) of the contacted PEgRNA, thereby priming DNA polymerization.
  • PBS primer binding site sequence
  • the DNA polymerization is reverse transcription catalyzed by a reverse transcriptase domain of the prime editor.
  • the method comprises contacting the target gene with a DNA polymerase, e.g., a reverse transcriptase, as a part of a prime editor fusion protein or prime editing complex (in cis), or as a separate protein (in trans).
  • a DNA polymerase e.g., a reverse transcriptase
  • contacting the target gene with the prime editing composition generates an overlap duplex (OD) or replacement duplex (RD) that replaces the IND.
  • the OD or RD comprises one or more intended nucleotide edits compared to the endogenous sequence of the target gene, e.g., a TRAC gene.
  • the intended nucleotide edits are incorporated in the target gene by replacement of the IND by the OD or RD. In some embodiments, the intended nucleotide edits are incorporated in the target gene by excision of the IND and DNA repair. [0609] In some embodiments, incorporation of the intended nucleotide edits in a TRAC gene results in disruption of the TRAC gene that reduces or abolishes expression of the gene.
  • the intended nucleotide edits comprise an insertion in the TRAC gene that disrupts the reading frame of the gene. In some embodiments, the intended nucleotide edits comprise an insertion that includes one or more stop codons.
  • the intended nucleotide edits comprise an insertion of one or more recombinase recognition sequences (RSSs), wherein the insertion disrupts the reading frame of the TRAC gene.
  • editing of a TRAC gene further comprises contacting the TRAC gene with one or more recombinases or one or more polynucleotides encoding the recombinases, wherein the PEgRNA or PEgRNA pair of the prime editing composition encode insertion of one or more recombinase recognition sequences (RSSs) in TRAC gene.
  • editing of a TRAC gene further comprises contacting the TRAC gene with a DNA polynucleotide for recombination with TRAC containing the RSS(s) introduced by prime editing.
  • the DNA polynucleotide comprises (a) a donor sequence and (b) one or more RSSs recognized by the recombinase.
  • disclosed herein is a method for integrating a donor sequence in a target gene, wherein the method comprises contacting the target gene with any one of the prime editing systems or compositions described herein.
  • a target gene e.g., the TRAC gene
  • the prime editor or polynucleotides encoding the prime editor
  • PEgRNAs or polynucleotides encoding the recombinases
  • WSGR Docket No.59761-772601 and optionally the DNA polynucleotide can result in inversion, deletion, insertion of or replacement with an exogenous donor sequence, and other recombination events.
  • a method of targeted insertion of an exogenous donor sequence into the TRAC gene comprises contacting the TRAC gene with (a) a PEgRNA or a pair of PEgRNAs capable of inserting a first RSS in the TRAC gene, or one or more polynucleotides encoding the PEgRNA, (b) a prime editor or one or more polynucleotides encoding the prime editor, (c) a recombinase that recognizes the first RSS, or one or more polynucleotides encoding the recombinase, and (d) a DNA polynucleotide comprising (i) a second RSS recognized by the recombinase and (ii) a donor sequence.
  • a method of targeted replacement of an endogenous TRAC sequence with an exogenous donor sequence comprises contacting the TRAC gene with (a) a PEgRNA or a pair of PEgRNAs capable of inserting a first RSS and a second RSS in the TRAC gene, or one or more polynucleotides encoding the PEgRNA, (b) a prime editor or one or more polynucleotides encoding the prime editor, (c) a recombinase that recognizes the first RSS, or one or more polynucleotides encoding the recombinase, and (d) a DNA polynucleotide comprising (i) a third RSS and a fourth RSS recognized by the recombinase and (ii) a donor sequence between the third and the fourth RSSs.
  • a method of targeted deletion of an endogenous sequence in the TRAC gene comprises contacting the TRAC (a) a PEgRNA or a pair of PEgRNAs capable of inserting a first RSS and a second RSS in the TRAC gene, or one or more polynucleotides encoding the PEgRNA, (b) a prime editor or one or more polynucleotides encoding the prime editor, and (c) a recombinase that recognizes the first and the second RSS.
  • a method of introducing a targeted inversion of an endogenous sequence in the TRAC gene comprises contacting the TRAC (a) a PEgRNA or a pair of PEgRNAs capable of inserting a first RSS and a second RSS in the TRAC gene, or one or more polynucleotides encoding the PEgRNA, (b) a prime editor or one or more polynucleotides encoding the prime editor, and (c) a recombinase that recognizes the first and the second RSS.
  • a method of targeted insertion of an exogenous donor sequence into the TRAC gene comprises contacting the TRAC gene with (a) a first PEgRNA comprising (i) a first spacer that is complementary to first search target sequence on a first strand of the TRAC gene, (ii) a first gRNA core capable of binding to a Cas9 protein, and (iii) a first extension arm comprising a first editing template and a first primer binding site (PBS), or one or more polynucleotides encoding the WSGR Docket No.59761-772601 first PEgRNA, (b) a second PEgRNA comprising (i) a second spacer that is complementary to second search target sequence on a second strand of the TRAC gene, (ii) a second gRNA core capable of binding to a Cas9 protein, and (ii
  • the first PEgRNA comprises a spacer selected from any one of the 5’ spacer sequences of Tables 9-12, and the first PBS comprises a PBS sequence selected from the same table as the first spacer.
  • the second PEgRNA comprises a spacer selected from any one of the 3’ spacer sequences of Tables 13-25, and the second PBS comprises a PBS sequence selected from the same table as the second spacer.
  • the first editing template encodes a first single stranded DNA
  • the second editing template encodes a second single stranded DNA
  • incorporation of the first and second single stranded DNA in the TRAC gene via dual prime editing results in replacement of the IND with a replacement duplex (RD) based on sequences of the first and second single stranded DNA, wherein the RD contains the first RSS sequence.
  • the first editing template encodes the first RSS sequence.
  • the second editing template encodes the first RSS sequence.
  • the first and second RSS sequences and the recombinase can be any RSS and corresponding recombinases known in the art.
  • the first RSS is an attB sequence
  • the second RSS is an attP sequence, or vice versa.
  • the recombinase is Bxb1.
  • the first editing template comprises a RTT#1 sequence in Table 6 having the RTT Pair No.1 and the second editing template comprises a RTT#2 sequence in Table 6 having the same RTT Pair No.
  • the first editing template comprises a RTT#2 sequence in Table 6 having the RTT Pair No.1 and the second editing template comprises a RTT#1 sequence in Table 6 having the same RTT Pair No.
  • the recombinase is Pa01.
  • the first editing template comprises a RTT#1 sequence in Table 6 having the RTT Pair No.2 or 3 and the second editing template comprises a RTT#2 sequence in Table 6 having the same RTT Pair No. In some embodiments, the first editing template comprises a RTT#2 sequence in Table 6 having the RTT Pair No.2 or 3 and the second editing template comprises a RTT#1 sequence in Table 6 having the same RTT Pair No.
  • the first editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the second editing template comprises a full length or 5’ fragment of the WSGR Docket No.59761-772601 corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the second editing template comprises a 5’ fragment of an RTT listed in Table 6 and wherein the first editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second editing templates have perfect reverse complementarity to each other.
  • the first PEgRNA contains a PEgRNA sequence selected from any one of Tables 9-12
  • the second PEgRNA contains a PEgRNA sequence selected from any one of Tables 13-25, wherein the second PEgRNA comprises an editing template that has the same RTT Pair Number as the first PEgRNA.
  • the DNA polynucleotide can be any suitable sequence or configuration, e.g., a plasmid or a minicircle.
  • the donor sequence in the DNA polynucleotide can be any sequence for targeted integration into the TRAC gene.
  • the donor sequence can comprise one or more expression cassettes containing one or more promoters.
  • the donor sequence comprises sequences that encode one or more polypeptides.
  • the donor sequence comprises one or more open reading frames (ORFs) that encode one or more polypeptides.
  • the polypeptide(s) can comprise a cellular receptor, an immune checkpoint protein, a cytokine, a chemokine, a dominant negative mutant of a protein involved in immune cell signaling, a transcription factor, a signaling protein, a reporter protein, an epitope tag, a kill switch, or any protein or functional portion or fragment thereof for integration into the TRAC gene as described herein or known in the art.
  • the method of editing the TRAC gene comprises contacting the TRAC gene with the PEgRNAs or polynucleotide(s) encoding same, the prime editor or polynucleotide(s) encoding same followed by contacting the TRAC gene with the recombinase or polynucleotide(s) encoding same, and the DNA polynucleotide.
  • the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after contacting the TRAC (e.g., by introducing into a cell containing the TRAC) with a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs.
  • the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided at the same time a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs.
  • the target TRAC gene is a wild-type TRAC gene.
  • the target TRAC gene comprises one or more mutations compared to a wild-type TRAC sequence.
  • the target gene e.g., a TRAC gene, is in a cell. Accordingly, also provided herein are methods of modifying, engineering, or editing a cell, such as a human cell, a human primary cell, a human iPSC-derived cell, and/or a human T-cell or a precursor thereof, or a progenitor thereof.
  • the prime editing method comprises introducing a first PEgRNA, a second PEgRNA, and a prime editor into the cell that has the target gene.
  • the method comprises further introducing into the cell a recombinase or one or more polynucleotide encoding the recombinase and optionally a DNA polynucleotide comprising one or more RSSs recognized by the recombinase and a donor sequence.
  • the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a first PEgRNA, a second PEgRNA, and a prime editor polypeptide.
  • the first PEgRNA, the second PEgRNA, and the prime editor polypeptides form complexes prior to the introduction into the cell. In some embodiments, the first PEgRNA, the second PEgRNA, and the prime editor polypeptides form complexes after the introduction into the cell. In some embodiments, the recombinase is fused, linked, or otherwise complexed with the prime editor polypeptide.
  • the prime editors, PEgRNAs, recombinases, DNA polynucleotides containing donor sequences, and prime editing complexes may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, including ribonucleoprotein (RNPs), lipid nanoparticles (LNPs), viral vectors, non-viral vectors, mRNA delivery, and physical techniques such as cell membrane disruption by a microfluidics device.
  • RNPs ribonucleoprotein
  • LNPs lipid nanoparticles
  • viral vectors non-viral vectors
  • mRNA delivery mRNA delivery
  • the prime editors, PEgRNAs, and prime editing complexes may be introduced into the cell simultaneously or sequentially.
  • the prime editing method comprises introducing into the cell a first PEgRNA and a second PEgRNA, or polynucleotides encoding the first PEgRNA and the second PEgRNA, and a prime editor polynucleotide encoding a prime editor polypeptide.
  • the method comprises introducing the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA, and the polynucleotide encoding the prime editor polypeptide into the cell simultaneously.
  • the method comprises introducing the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA, and the polynucleotide encoding the prime editor polypeptide into the cell sequentially. In some embodiments, the method comprises introducing the polynucleotide encoding the prime editor polypeptide into the cell before introduction of the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA.
  • the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA are introduced into the cell.
  • the method further comprises introducing into the cell a recombinase or one or more polynucleotides encoding the recombinase. In some embodiments, the method further comprises introducing into the cell a DNA polynucleotide comprising one or more RSSs recognized by the recombinase and a donor sequence. In some embodiments, the recombinase and/or the one or more polynucleotides encoding the recombinase are introduced after introduction of first PEgRNA, second PEgRNA, prime editor, or the one or more polynucleotides encoding the same.
  • the recombinase and/or the one or more polynucleotides encoding the recombinase are introduced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after introduction of first PEgRNA, second PEgRNA, prime editor, or the one or more polynucleotides encoding the same.
  • the one or more polynucleotides encoding the PEgRNAs, the one or more polynucleotides encoding the prime editor, and/or the one or more polynucleotides encoding the recombinase are in DNA vectors, e.g., AAV vectors.
  • one or more polynucleotides encoding the prime editor and/or the one or more polynucleotides encoding the recombinase are mRNA.
  • the one or more polynucleotide encoding the prime editor polypeptide, the first PEgRNA and the second PEgRNA or the polynucleotides encoding the first PEgRNA and the second PEgRNA, the recombinase or the one or more polynucleotides encoding the recombinase, and the DNA polynucleotide may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, for example, by RNPs, LNPs, viral vectors, non-viral vectors, mRNA delivery, and physical delivery.
  • the polynucleotide encoding the prime editor polypeptide and the polynucleotides encoding the first PEgRNA and the second PEgRNA integrate into the genome of the cell after being introduced into the cell.
  • the polynucleotide encoding the prime editor polypeptide and the polynucleotides encoding the first PEgRNA and the second PEgRNA are introduced into the cell for transient expression.
  • the one or more polynucleotides encoding the recombinase are introduced into the cell for transient expression.
  • the cell is a mammalian cell. In some embodiments, the cell is a non- human primate cell, bovine cell, porcine cell, rodent, or mouse cell. In some embodiments, the cell is a human cell. In some embodiments, the edited cell (or the population of edited cells) is a mammalian cell. In some embodiments, the edited cell is a human cell. In some embodiments, the cell is an WSGR Docket No.59761-772601 immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the cell is a T cell, a B cell, a NK cell, or a progenitor or a precursor thereof.
  • the cell is a T cell, a B cell, a NK cell, or a progenitor or a precursor thereof.
  • the cell is a primary cell. In some embodiments, the cell is a human primary cell. [0631] In some embodiments, the target gene, e.g., the TRAC gene, edited by prime editing is in a chromosome of the cell. In some embodiments, the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells. In some embodiments, the intended nucleotide edits introduced to the cell by the prime editing systems, compositions, and methods are such that the cell and progeny of the cell also include the intended nucleotide edits. In some embodiments, the cell is autologous, allogeneic, or xenogeneic to a subject.
  • the cell is from or derived from a subject. In some embodiments, the cell is from or derived from a human subject. In some embodiments, the cell is introduced back into the subject, e.g., a human subject, after incorporation of the intended nucleotide edits by prime editing.
  • the method provided herein comprises introducing the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, and the PEgRNAs (e.g., the first PEgRNA and the second PEgRNA) or the polynucleotides encoding the PEgRNAs, into a plurality or a population of cells that comprise the target gene.
  • the population of cells is of the same cell type. In some embodiments, the population of cells is of the same tissue or organ. In some embodiments, the population of cells is heterogeneous. In some embodiments, the population of cells is homogeneous. In some embodiments, the population of cells is from a single tissue or organ, and the cells are heterogeneous. In some embodiments, the introduction into the population of cells is ex vivo. In some embodiments, the introduction into the population of cells is in vivo, e.g., into a human subject. [0633] In some embodiments, the target gene, e.g., the TRAC gene, is in a genome of each cell of the population.
  • the target gene e.g., the TRAC gene
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, and the PEgRNAs (e.g., the first PEgRNA and the second PEgRNA) or the polynucleotides encoding the PEgRNAs results in incorporation of one or more intended nucleotide edits in the target gene in at least one of the cells in the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, and the PEgRNAs or the polynucleotides encoding the PEgRNAs results in incorporation of the one or more intended nucleotide edits in the target gene in a plurality of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, and the PEgRNAs or the polynucleotide encoding the PEgRNAs, results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells.
  • incorporation of the intended nucleotide edits in a TRAC gene in a plurality or population of cells results in disruption of the TRAC WSGR Docket No.59761-772601 gene that reduces or abolishes expression of the gene.
  • the intended nucleotide edits comprise an insertion in the TRAC gene that disrupts the reading frame of the gene.
  • the intended nucleotide edits comprise an insertion that includes one or more stop codons.
  • the intended nucleotide edits comprise an insertion of one or more recombinase recognition sequences (RSSs), wherein the insertion disrupts the reading frame of the TRAC gene.
  • RSSs recombinase recognition sequences
  • incorporation of the intended nucleotide edits results in disruption of the TRAC gene and reduced or abolished expression of the TRAC gene in a fraction of the population of cells or in each cell of the population of cells.
  • the method provided herein further comprises introducing a recombinase or one or more polynucleotides encoding the recombinase, and optionally a DNA polynucleotide comprising one or more RSSs recognized by the recombinase and a donor sequence into a plurality or a population of cells that comprise the TRAC gene.
  • Introduction of the recombinase or the polynucleotide(s) encoding the recombinase and the DNA polynucleotide can be performed simultaneously or sequentially (e.g. after) introduction of the prime editor or polynucleotide(s) encoding the prime editor and the PEgRNAs or polynucleotides encoding the PEgRNAs.
  • introduction of the recombinase or one or more polynucleotides encoding the recombinase the DNA polynucleotide results in integration of the donor sequence into the TRAC gene.
  • Disruption of the TRAC gene and reduced/abolished expression of the CD3 protein may be therapeutically relevant in immune therapy.
  • immune therapy using allogeneic immune cells e.g., T cells from healthy donors
  • GvHD graft-versus-host-disease
  • Reduced or abolished expression of CD3 disrupts the endogenous T cell receptor (TCR) complex, which may lead to reduced GvHD and allow for wide clinical application of allogeneic T cells.
  • the donor sequence can comprise any transgene or open reading frame (ORF) that is therapeutically relevant.
  • the donor sequence comprises a sequence, e.g., an ORF that encodes a polypeptide that is involved in signaling, binding, regulation, or activity of an immune cell, e.g., a T cell.
  • the polypeptide may be a cellular receptor, an immune checkpoint protein, a cytokine, a chemokine, a dominant negative mutant of a protein involved in immune cell signaling, a transcription factor, a signaling protein, a reporter protein, an epitope tag, a kill switch, or any protein or functional portion or fraction thereof.
  • the polypeptide may thus function to direct the immune cell to a specific target, e.g., a tumor cell, activate the immune cell, reduce tumor inhibition of immune response, regulate immune cell activity, or selectively eliminate certain immune cells.
  • the polypeptides encoded by the donor sequence in combination with disruption of the TRAC gene can be used for generation of WSGR Docket No.59761-772601 allogeneic immune cells, e.g., T cells, having improved target specificity (e.g., tumor specificity mediated by CARs or surface receptors), improved activity, reduced GvHD effect, and/or regulation potential in immune therapy.
  • target specificity e.g., tumor specificity mediated by CARs or surface receptors
  • improved activity e.g., tumor specificity mediated by CARs or surface receptors
  • reduced GvHD effect e.g., reduced GvHD effect
  • regulation potential in immune therapy e.g., provided herein are methods of editing a cell or population of cells and cells edited by prime editing.
  • pharmaceutical compositions comprising a prime editing composition described herein, for example, a pharmaceutical composition comprising a LNP comprising a prime editing composition (or components thereof) described herein.
  • a disease e.g., a cancer
  • the method comprises administering the pharmaceutical compositing a prime edited cell or a population of prime edited cells to the subject.
  • an isolated cell or a population of cells e.g., an isolated T cell or a population of T cells generated by the prime editing methods disclosed herein.
  • the edited cell or population of cells comprise a disruption in the TRAC gene, wherein the disruption reduces or abolishes expression or function of the CD3 protein.
  • the edited cell or population of cells comprises an exogenous sequence (i.e. the integrated donor sequence) in the TRAC gene.
  • the exogenous sequence comprises an expression cassette comprising a gene or an open reading frame (ORF) driven by one or more promoters.
  • the exogenous sequence comprises multiple expression cassettes each driven by one or more promoters.
  • the exogenous sequence comprises an expression cassette comprising two or more genes or ORFs driven by a promoter, wherein the two or more genes or ORFs are separated by a sequence encoding a self-cleaving peptide, e.g., a 2A peptide.
  • the exogenous sequence does not contain a promoter, but contains a splice acceptor sequence preceding the start codon of the transgene ORF.
  • a transgene or transgene ORF in the donor sequence encodes a polypeptide.
  • the polypeptide is involved binding, regulation, or activity of an immune cell, e.g., a T cell, and may function in combination or synergistically with disruption of the TRAC gene by prime editing.
  • the polypeptide is a cellular receptor or a portion thereof, e.g., a receptor that directs specific antigen binding.
  • the polypeptide may be a chimeric antigen receptor (CAR), a T cell receptor (TCR), a B cell receptor, a NK cell receptor, or a functional portion thereof.
  • the CAR has specificity for a target antigen that is a disease associated antigen.
  • the CAR specifically binds a target antigen or a neoantigen expressed by a cancer cell (e.g., expressed or presented on the surface of a cancer cell).
  • the target antigen or a neoantigen is from an oncogene or tumor suppressor gene (e.g., a mutated tumor suppressor gene).
  • the target antigen comprises a T cell epitope.
  • the target antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide.
  • the target antigen is autologous, and is expressed on a diseased or otherwise altered cell.
  • the target antigen is expressed in a neoplastic cell.
  • the CAR or cellular receptor specifically binds to a target antigen expressed by a pathogen e.g., a bacterium, virus, fungus, yeast, parasite (e.g., single-celled or multicellular eukaryotic parasite), or other microorganism.
  • the cellular receptor or CAR specifically binds to a target antigen associated with a disease (e.g., an inflammatory or autoimmune disease).
  • Exemplary CARs include a CD19 CAR, CD20 CAR, BAFF CAR, B7H2 CAR, GD2 CAR, MSLN CAR, HER2 CAR, EGFR CAR, CD70 CAR, BCMA CAR, or other CARs that are therapeutically relevant.
  • Exemplary TCRs include NY-ESO-1, AHNAK(S2580F), ERBB2(H473Y), HPV-E6, MAGE-A3/A6, E7, HA-1, or other TCRs that are therapeutically relevant.
  • Exemplary NK cell receptors CD16, NKG2D, DNAM1, NKp46, NKp44, NKp30, LFA1, CD27, or other NK cell receptors that are therapeutically relevant.
  • the polypeptide is a cellular receptor or a functional portion thereof, e.g., a receptor that modulates immune cell activity.
  • the polypeptide is a cytokine receptor, e.g., IL2RA, IL2RB, IL7R, IL12R, IL18R, or IFNGR, or a functional portion thereof.
  • the polypeptide is a chemokine receptor, e.g., CCR2, CCR4, CCR5, CCR6, CXCR1, CXCR2, CXCR3, CXCR4, or a functional portion thereof.
  • the polypeptide is an immune checkpoint protein, e.g., PD1, CTLA4, CISH, TIGIT, TIM3, or LAG3.
  • the polypeptide is a cytokine, e.g., IL2, IL15, IL7, TNF, or IFNG.
  • the polypetide is a chemokine, e.g., CCL2, CCL5, CCL17, CCL20, CCL22, CXCL2, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11, or CXCL12.
  • the polypeptide is a dominant-negative mutant of a protein involved in immune cell signaling, e.g.
  • the polypeptide may be a dominant negative mutant of TGFBR, IL10R, IL35R, FOXP3, or STAT5.
  • the polypeptide is a transcription factor involved in immune cell signaling, e.g. T cell signaling.
  • the polypeptide may be BLIMP1, IL2RA, IL2RB, BCL6, ID2, C-MYB, IL6R, TCF1, EOMES, STAT1, STAT3, STAT4, STAT5, TBET, NFkB, GATA-3, IRF-4.
  • the polypeptide may be a signaling protein, e.g., LCK, SYK, or ZAP70.
  • the polypeptide is a reporter protein, e.g., a fluorescence protein.
  • the polypeptide is an epitope tag.
  • the polypeptide is a “suicide protein” or a “kill switch” that allows for specific elimination of cells containing the polypeptide, e.g., for specific elimination of edited cells after administration.
  • the polypeptide may be iCasp9, HSV-TK, or an epitope for antibody-mediated elimination (e.g., CD20 for rituximab-mediated elimination).
  • the edited cell or population of cells comprises a sequence in the TRAC gene resulting from recombination between a first RSS in the TRAC gene inserted by prime editing and a second RSS in an exogenous DNA polynucleotide provided.
  • the WSGR Docket No.59761-772601 first RSS is an attP sequence and the second RSS is an attB sequence.
  • the sequence resulted from the recombination comprises an attL sequence and an attR sequence.
  • the edited cell or population of cells are contacted with the recombinase Bxb1.
  • the edited cell or population of cells comprise an attL sequence GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1046) and/or an attR sequence GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1047).
  • the edited cell or population of cells further comprise one or more mutations in an endogenous gene that result in reduced or abolished expression of the endogenous gene.
  • the edited cell or population of cells further comprises one or more mutations in each of multiple endogenous genes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes, that result in reduced or abolished expression of the multiple endogenous genes.
  • Mutations in the endogenous gene(s) may be introduced by prime editing or dual prime editing methods as described herein, or by other gene editing approaches such as programmable nuclease mediated knockdown or knockout, e.g., by CRISPR-Cas9 nuclease, zinc finger nuclease, or TALEN designed for specific cleavage of the endogenous gene sequence(s).
  • Such endogenous genes may be involved in specificity, function, regulation, and/or activity of an immune cell.
  • the endogenous gene encodes a cellular receptor, or a component of a cellular receptor signaling pathway, e.g., other components of the TCR complex.
  • the endogenous gene encodes a human leukocyte antigen (HLA) component, for example, ⁇ 2-microglobulin (B2M). Similar to disruption of the TRAC gene, knockout of other TCR complex components or HLA components may also reduce GvHD effect and improve allogeneic immune cells used in cell therapy.
  • the endogenous gene encodes a checkpoint protein, for example, PDCD1. Knockout of checkpoint proteins may reduce tumor inhibition of immune cell activity, thereby improve on-tumor activity of the immune cells used in cell therapy.
  • the endogenous genes include CD2, CD3 epsilon, CD3 gamma, CD3 delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and CIITA.
  • the edited cell or population of cells further comprise one or more transgenes that express one or more functional proteins.
  • the one or more transgenes may be introduced into a specific genomic location by prime editing and recombinase mediated integration methods as described herein, or by other gene editing approaches such as programmable nuclease directed homologous recombination (HDR), e.g., by CRISPR-Cas9 nuclease, zinc finger nuclease, or TALEN combined with HDR donors.
  • HDR programmable nuclease directed homologous recombination
  • the transgenes may also be introduced non-specifically into the cell, e.g., by lentivirus.
  • the transgenes may encode any protein.
  • the polypeptide may be a cellular receptor, an immune checkpoint protein, a cytokine, a chemokine, a dominant negative mutant of a protein involved in immune cell signaling, a transcription factor, a signaling protein, a WSGR Docket No.59761-772601 reporter protein, an epitope tag, a kill switch, or any therapeutic protein or functional portion or fragment thereof.
  • the edited cells exhibit reduced the alloreactivity, and may prevent graft vs host disease upon administration to a subject in need. In some embodiments, the edited cells exhibit reduced alloreactivity as compared to a suitable control cell.
  • administering an effective amount of a composition comprising a population of the edited cells of the disclosure induces a reduced alloreactivity (e.g., by at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more) to the edited cells in the subject compared to that induced upon administering of composition comprising a population of suitable control cells.
  • a reduced alloreactivity e.g., by at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more
  • methods to reduce alloreactivity to a cell comprising contacting a cell with an effective amount of a prime editing composition disclosed herein.
  • the edited cells are non-alloreactive cells.
  • the edited cell (or the population of edited cells) is a mammalian cell. In some embodiments, the edited cell is a human cell. In some embodiments, the cell is an immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the cell is a T cell, a B cell, a NK cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a human T cell, or a progenitor or a precursor thereof.
  • an immune cell e.g., a primary immune cell
  • the cell is a T cell, a B cell, a NK cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a human T cell, or a progenitor or a precursor thereof.
  • the cell is a T cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T cell, a CD4+ T cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de-differentiated cell thereof, and stem cells.
  • a primary T cell e.g., an inflammatory T cell, a T helper cell, a cytotoxic T cell, a CD4+ T cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a
  • a cell is a naive T cell (e.g., a naive CD8+ T cell).
  • the cell is a transformed T cell.
  • the cell is a memory T cell (e.g., (e.g., central memory T cell (TCM), stem memory T cell (TSCM), effector memory T cell, Tissue resident memory T cell).
  • the cell is an effector memory T cell (e.g., TEM cells and TEMRA (CD45RA+) cells).
  • the cell is a regulatory T cell.
  • the cell is a natural killer T cell.
  • the cell is a Mucosal associated invariant T cell.
  • the cell is a T cell. In some embodiments, the cell is an effector T cell. In some embodiments, the cell is a T helper cell (e.g., Th1 cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, and Tfh (follicular helper) cell). In some embodiments, the cell is a thymocyte. In some embodiments, the cell is a lymphoid cell. In some embodiments, the cell is a common lymphoid progenitor cells. In some embodiments, the cell is an early thymic progenitor cell. In some embodiments, the cell is a CD3+ cell. In some embodiments, the cell is a tumor infiltrating lymphocyte.
  • T helper cell e.g., Th1 cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, and Tfh (follicular helper) cell.
  • the cell is a thymocyte.
  • the cell is a lymphoid cell.
  • the cell is a myeloid cell. In some embodiments, the cell is a plasma cell. In some embodiments, the cell is an activated T cell. WSGR Docket No.59761-772601 [0645] In some embodiments, the cell is a progenitor cell. In some embodiments, the cell is a human progenitor cell. In some embodiments, the cell is a stem cell (e.g., adult stem cell, embryonic stem cell, non-embryonic stem cell), cord blood stem cell, progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell).
  • stem cell e.g., adult stem cell, embryonic stem cell, non-embryonic stem cell
  • cord blood stem cell progenitor cell
  • progenitor cell bone marrow stem cell
  • induced pluripotent stem cell induced pluripotent stem cell
  • totipotent stem cell a
  • the cell is a pluripotent cell (e.g., a pluripotent stem cell).
  • the cell e.g., a stem cell
  • the cell is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell.
  • the cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the cell is a hematopoietic stem cell.
  • the cell is a hematopoietic stem and progenitor cell.
  • the cell is a multipotent progenitor cell.
  • the cell is a T-cell progenitor.
  • the cell is a T-cell precursor. In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human pluripotent stem cell. In some embodiments, the cell is a non-embryonic stem cell. In some embodiments, the cell is an induced human pluripotent stem cell. In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human T- cell progenitor. In some embodiments, the cell is a human T-cell precursor.
  • ESC embryonic stem cell
  • the cell is a human stem cell.
  • the cell is a human pluripotent stem cell.
  • the cell is a non-embryonic stem cell.
  • the cell is an induced human pluripotent stem cell.
  • the cell is a human stem cell.
  • the cell is a human embryonic stem cell.
  • the cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal.
  • the cell is a differentiated cell.
  • the cell is differentiated from an induced pluripotent stem cell.
  • the cell is a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell differentiated from an iPSC, ESC, a T-cell precursor, or a T-cell progenitor.
  • the cell is a differentiated human cell.
  • the cell is differentiated from an induced human pluripotent stem cell.
  • the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T- cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a ⁇ T cell, an alpha beta T cell, a naive T cell, or an effector T cell.
  • a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T- cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T
  • the cell is in a subject, e.g., a human subject.
  • the cell is obtained from a subject prior to editing.
  • the cell is obtained from a patient having a cancer, a microbial infection, a graft vs host disease, or an autoimmune disorder.
  • the cell is obtained from one or more healthy donors.
  • the cell Prior to editing by the methods and compositions disclosed herein, the cell can be obtained from a subject through a variety of non-limiting methods.
  • T cells can be obtained from a number of non- WSGR Docket No.59761-772601 limiting sources, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • Methods of collecting blood cells, isolating and enriching T cells, and expanding them ex vivo may be by methods known in the art.
  • the cell can be obtained from a cell bank, a blood bank, cell culture, or any number of T cell lines available, and known to those skilled in the art. Cells may also be obtained from a tissue biopsy, surgery, blood, plasma, serum, or other biological fluid.
  • the cell can be obtained prior to editing from one or more healthy donors, from a patient having a cancer, a microbial infection, a graft versus host infection, or an autoimmune disorder.
  • the edited cells are expanded ex vivo prior to administering to a subject.
  • the edited cells are expanded in vivo, for example, after administering the edited cells to a subject.
  • the edited cells can be used as a medicament.
  • the medicament can be used to treat subjects having, diagnosed with, or at risk of developing a disease, disorder, or a condition, e.g., a cancer, a microbial infection such as a pathogenic infection, a bacterial infection, a viral infection, an autoimmune disorder, or a graft versus host disease.
  • the edited cells can be used in manufacture of a medicament for a treatment of a disease, disorder, or a condition, e.g., cancer, a microbial infection, such as a pathogenic infection, a bacterial infection, a viral infection, an autoimmune disorder, or a graft versus host disease.
  • the subject can be undergoing or in need of an immunosuppressive therapy.
  • provided herein are methods of treatment of a subject in need thereof. In one aspect, provided herein are methods of treatment or prevention of a disease, disorder, or condition in a subject.
  • the subject is diagnosed with, has, or is at a risk of developing a disease, disorder, or a condition.
  • the subject is in need of, or undergoing, or will be undergoing an immunotherapy, for example, an autologous immune cell immunotherapy, an allogenic immune cell immune therapy.
  • the disease, disorder, or a condition is responsive to immunotherapy, for example, an autologous immune cell immunotherapy, an allogenic immune cell immune therapy.
  • the methods disclosed herein comprise administering an effective amount of a prime editing composition disclosed herein, the edited cells disclosed herein, or a composition comprising the edited cells disclosed herein to the subject.
  • Editing efficiency [0652] In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition.
  • the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a TRAC gene within the genome of a WSGR Docket No.59761-772601 cell) to a prime editing composition.
  • a target gene e.g., a TRAC gene within the genome of a WSGR Docket No.59761-772601 cell
  • editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition.
  • the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of exposing a target gene (e.g., a TRAC gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a TRAC gene within the genome of a cell
  • the population of cells introduced with the prime editing composition is ex vivo.
  • the population of cells introduced with the prime editing composition is in vitro.
  • the population of cells introduced with the prime editing composition is in vivo.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% relative to a suitable control.
  • the prime editing methods disclosed herein have an editing efficiency of at least 25% relative to a suitable control.
  • the prime editing methods disclosed herein have an editing efficiency of at least 35% relative to a suitable control prime editing method disclosed herein has an editing efficiency of at least 30% relative to a suitable control.
  • the prime editing methods disclosed herein have an editing efficiency of at least 45% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 50% relative to a suitable control. In some embodiments, editing efficiency of prime the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells after in vivo engraftment of the edited cells. In some embodiments, the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of engraftment. In some embodiments, the editing efficiency is determined after 8 or 16 weeks of engraftment.
  • prime editing is able to maintain in edited cells at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more than 95% of editing efficiency after 8 or 16 weeks post engraftment.
  • the methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a primary cell relative to a suitable control.
  • the methods disclosed herein have an editing efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a target cell (e.g., a human primary WSGR Docket No.59761-772601 cell, human iPSC, or human T cell or a progenitor or a precursor thereof) relative to a corresponding control target cell.
  • a target cell e.g., a human primary WSGR Docket No.59761-772601 cell, human iPSC, or human T cell or a progenitor or a precursor thereof
  • the target cell is a human cell (e.g., a human primary cell, human iPSC, or human T cell, or a progenitor or a precursor thereof).
  • the methods disclosed herein results in integration of a donor sequence at an efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a target cell or target cell population (e.g., a human primary cell, human iPSC, or human T cell or a progenitor or a precursor thereof) relative to a corresponding control target cell.
  • a target cell or target cell population e.g., a human primary cell, human iPSC, or human T cell or a progenitor or a precursor thereof
  • the target cell is a human cell (e.g., a human primary cell, human iPSC, human immune cell, or human T cell, or a progenitor or a precursor thereof).
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits without generating a significant proportion of indels.
  • the term “indel(s)”, as used herein, refers to the insertion or deletion of a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. Indel frequency of editing can be calculated by methods known in the art.
  • indel frequency can be calculated based on sequence alignment such as the CRISPResso 2 algorithm as described in Clement et al., Nat. Biotechnol.37(3): 224-226 (2019), which is incorporated herein in its entirety.
  • the methods disclosed herein can have an indel frequency of less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, or less than 1%.
  • any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a TRAC gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a TRAC gene within the genome of a cell
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits efficiently without generating a significant proportion of indels.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or muscle WSGR Docket No.59761-772601 cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.
  • any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a TRAC gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a TRAC gene within the genome of a cell
  • the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a TRAC gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a TRAC gene within the genome of a cell
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits efficiently without generating a significant proportion of indels.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a population of target cells. [0671] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an WSGR Docket No.59761-772601 indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a population of target cells. [0673] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a population of target cells.
  • WSGR Docket No.59761-772601 [0674] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a population of target cells. [0675] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing WSGR Docket No.59761-772601 methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a population of target cells. [0677] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a population of target cells. [0678] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a population of target cells. [0679] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 10% in a population of target cells. In some WSGR Docket No.59761-772601 embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a population of target cells. [0681] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than WSGR Docket No.59761-772601 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a population of target cells. [0682] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% as measured in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90%.
  • the population of target cell comprises a population of human T-cells.
  • the prime editing composition described herein results in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in a chromosome that includes the target gene.
  • off-target editing is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a nucleic acid within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a nucleic acid within the genome of a cell
  • the prime editing methods described herein result in less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 4% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 3% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 2% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 1%large deletion in edited cells.
  • the prime editing methods described herein does not result in detectable level of large deletion in edited cells.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene decreases expression and/or function of a TRAC protein.
  • expression and/or function of a TRAC protein may be measured when expressed in a target cell.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene leads to a fold change in a level of TRAC gene expression, TRAC protein expression, TRAC protein function, or a combination thereof.
  • a change in the level of TRAC gene expression, TRAC protein expression, TRAC protein function, or a combination thereof can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold or more as compared to expression in a suitable control cell.
  • a change in the level of a TRAC protein function can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to expression in a suitable control cell.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene decreases expression of a TRAC gene, TRAC mRNA and/or TRAC protein e.g., by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more as compared to the expression in a suitable control cell.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene results in abolished or complete loss of expression of a TRAC gene, and/or a TRAC protein as compared to the expression in a suitable control cell.
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene decreases function of TRAC protein e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target TRAC gene results in abolished or complete loss of function of the TRAC protein as compared to that in a suitable control cell.
  • the function of a TRAC protein comprises associating with a T cell receptor B chain, associating with one or more CD3 ⁇ , ⁇ , ⁇ , and ⁇ chains and/or expression of a TCR (e.g., a functional TCR) on the surface of a cell.
  • a TCR e.g., a functional TCR
  • incorporation of the one or more intended nucleotide edits in the target TRAC gene increases expression of a truncated TRAC protein e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to expression of a full length wild type TRAC protein.
  • a suitable control cell is a cell that comprises a wild type TRAC gene, or is a cell not introduced or contacted with a prime editing composition described herein.
  • a suitable control cell is a cell from a healthy subject.
  • components of a prime editing composition described herein are provided to a target cell in vitro.
  • components of a prime editing composition described herein are provided to a target cell ex vivo. In some embodiments, components of a prime editing composition described herein are provided to a target cell in vivo. [0690] In some embodiments, incorporation of the one or more intended nucleotide edits in the target TRAC gene that reduces or abolishes expression and function of the protein encoded by the endogenous TRAC gene. [0691] In some embodiments, a decrease in expression and/or function of proteins of the TRAC gene can be measured by a functional assay. In some embodiments, expression and/or function of a exogenous protein encoded by a donor sequence introduced by prime editing and recombination can be measured by a functional assay.
  • a protein expression change can be measured by a protein expression assay.
  • the TRAC protein expression can be measured using antibody testing.
  • an antibody can comprise anti- TRAC.
  • the TRAC protein expression can be measured using ELISA, mass spectrometry, Western blot, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), electrophoresis, or any combination thereof.
  • a protein assay can comprise SDS-PAGE and densitometric analysis of a Coomassie Blue-stained gel.
  • WSGR Docket No.59761-772601 the method provided herein comprises administering to a subject an effective amount of a prime editing composition or a pharmaceutical composition comprising a prime editing composition described herein (i.e., for in vivo T cell editing).
  • the method comprises administering to a subject an effective amount of an LNP comprising a prime editing composition described herein.
  • the method provided herein comprises administering to a subject an effective amount of a pharmaceutical composition comprising a prime edited cell or a population of prime edited cells described herein.
  • the method comprises directly administering prime editing compositions provided herein to a subject, e.g., using a LNP that targets T cells and comprises a prime editing composition, e.g., a pair of dual prime editing PEgRNAs, an mRNA encoding a prime editor, and optionally (i) an mRNA encoding a recombinase that recognizes a RSS inserted by the PEgRNAs, and (ii) a DNA polynucleotide comprising a donor sequence and one or more RSSs recognized by the recombinase.
  • a prime editing composition e.g., a pair of dual prime editing PEgRNAs, an mRNA encoding a prime editor, and optionally (i) an mRNA encoding a recombinase that recognizes a RSS inserted by the PEgRNAs, and (ii) a DNA polynucleotide comprising a donor sequence and one or more RSSs recognized by the recombin
  • the pharmaceutical compositions can be administered to, e.g., a human subject in need of immune therapy, for example a human subject suffering from, having, susceptibility to, or at risk for a disease or condition, e.g., a cancer, a microbial infection, an autoimmune disorder, or a graft vs host disease. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).
  • the method comprises directly administering prime editing compositions provided herein to a subject.
  • the prime editing compositions described herein can be delivered, e.g., as LNPs.
  • the prime editing compositions can be formulated with any pharmaceutically acceptable carrier described herein or known in the art for administering directly to a subject.
  • Components of a prime editing composition or a pharmaceutical composition thereof may be administered to the subject, contacted to a TRAC gene, or introduced in a cell comprising a TRAC gene simultaneously or sequentially.
  • the method comprises administering, contacting with, or introducing a prime editing composition, or pharmaceutical composition thereof, comprising prime editor complexes that comprises (i) a prime editor fusion protein and a first PEgRNA and (ii) a prime editor fusion protein and a second PEgRNA to a subject.
  • the method comprises administering, contacting with, or introducing a polynucleotide or vector encoding a prime editor to a subject simultaneously with the two PEgRNAs. In some embodiments, the method comprises administering, contacting with, or introducing a polynucleotide or vector encoding a prime editor to a subject before administration of the two PEgRNAs. In some embodiments, the two PEgRNAs are administered simultaneously. In some embodiments, the two PEgRNAs are administered sequentially. In some embodiments, a first PEgRNA is administered with a prime editor and a second PEgRNA is administered after administration of the first PEgRNA and prime editor.
  • a first PEgRNA is WSGR Docket No.59761-772601 administered with a prime editor and a second PEgRNA is administered before administration of the first PEgRNA and prime editor.
  • Suitable routes of administrating the prime editing compositions to a subject include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
  • the compositions described are administered intraperitoneally, intravenously, or by direct injection or direct infusion. In some embodiments, the compositions described are administered by direct injection into the muscle of a subject. In some embodiments, the compositions described herein are administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant. [0697] In some embodiments, the method comprises to a subject an effective amount of the edited cells (e.g., a population of edited cells) or a composition comprising the edited cells (e.g., a population of edited cells) described herein (e.g., edited cells that are generated by the methods of the disclosure. In some embodiments, the edited cells are allogeneic.
  • the edited cells are allogeneic.
  • allogeneic cells are or have been contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are introduced into a subject (e.g., a human subject) in need thereof.
  • the edited cells are autologous to the subject.
  • cells are removed from a subject and contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are re- introduced into the subject.
  • cells are contacted ex vivo with one or more components of a prime editing composition.
  • the ex vivo-contacted cells are introduced into the subject, and the subject is administered in vivo with one or more components of a prime editing composition.
  • cells are contacted ex vivo with a prime editor and introduced into a subject.
  • the subject is a human subject.
  • the subject is then administered with the PEgRNAs, or polynucleotides encoding the PEgRNAs.
  • the ex vivo contacted cells are allogenic.
  • the ex vivo contacted cells are autologous.
  • cells contacted with the prime editing composition are determined for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject.
  • the cells are enriched for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject.
  • the cells of the disclosure are obtained from a subject in need of an immune cell immunotherapy (e.g., a CAR-T cell therapy) prior to ex vivo editing.
  • the autologous cells obtained from a subject prior to editing are cultured prior to WSGR Docket No.59761-772601 editing.
  • the autologous cells obtained from a subject for ex vivo prime editing are cultured, and edited shortly after they are obtained.
  • the autologous cells obtained from a subject and stored for future use e.g., future prime editing.
  • the edited cells are allogenic to the subject.
  • the prime editing composition or components thereof may be introduced into a cell by any delivery approaches as described herein, including LNP administration, RNP administration, electroporation, nucleofection, transfection, viral transduction, microinjection, cell membrane disruption and diffusion, or any other approach known in the art.
  • the cells edited with prime editing can be introduced into the subject by any route known in the art.
  • the edited cells are administered to a subject by direct infusion.
  • the edited cells are administered to a subject by intravenous infusion.
  • the edited cells are administered to a subject as implants.
  • the pharmaceutical compositions, prime editing compositions, and cells, as described herein, can be administered in effective amounts.
  • the effective amount depends upon the mode of administration. In some embodiments, the effective amount depends upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner.
  • the specific dose administered can be a uniform dose for each subject. Alternatively, a subject’s dose can be tailored to the approximate body weight of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient.
  • the time between sequential administration can be at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days.
  • a method of monitoring treatment progress is provided.
  • the method includes the step of determining a level of diagnostic marker, for example, expression of T-cell receptor, expression of a transgene, or measurement associated with a change in T-cell function and/or activity, in a subject who has been administered an effective amount of a prime editing composition or prime edited cells described herein.
  • the level of the diagnostic marker determined in the method can be compared to known levels of the marker in either healthy normal controls or in other afflicted subjects to establish the subject’s disease status.
  • kits comprising a prime editing composition.
  • a kit comprises a prime editing composition comprising a pair of PEgRNAs (i.e., a first PEgRNA and a second PEgRNA) and a prime editor.
  • a kit comprises a prime WSGR Docket No.59761-772601 editing composition comprising a pair of PEgRNAs (i.e., a first PEgRNA and a second PEgRNA) and a prime editor fusion protein.
  • the kit comprises a pair of PEgRNAs (i.e., a first PEgRNA and a second PEgRNA) and a polynucleotide encoding a prime editor.
  • the kit comprises a pair of PEgRNAs (i.e., a first PEgRNA and a second PEgRNA) and a polynucleotide encoding a prime editor fusion protein.
  • the kit further provides components for delivery of the PEgRNAs and/or the prime editor. In some embodiments, the kit further provides components for delivery of the PEgRNAs and/or the prime editor fusion protein. In some embodiments, the kit further provides components for delivery of the PEgRNAs and/or the polynucleotide encoding the prime editor. In some embodiments, the kit further provides components for delivery of the PEgRNAs and/or the polynucleotide encoding the prime editor fusion protein. [0708] In some embodiments, the kit provides instructions for using the components of the kit for prime editing. The instructions will generally include information about the use of the kit for editing nucleic acid molecules.
  • the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters.
  • the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.
  • the kit can further comprise a second container comprising a pharmaceutically acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Delivery [0709] Prime editing compositions described herein can be delivered to a cellular environment with any approach known in the art. Components of a prime editing composition can be delivered to a cell by the same mode or different modes.
  • a pharmaceutically acceptable buffer such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Delivery [0709] Prime editing compositions described herein can be delivered to
  • a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide.
  • a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA.
  • a prime editing composition component is encoded by a polynucleotide, a vector, or a construct.
  • a prime editor polypeptide and PEgRNAs is encoded by a polynucleotide.
  • the polynucleotide encodes a prime editor fusion protein comprising a DNA binding domain and a DNA polymerase domain.
  • the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a DNA binding domain of a prime editor. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a N- WSGR Docket No.59761-772601 terminal portion of a prime editor fusion protein connected to an intein-N. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a C-terminal portion of a prime editor fusion protein connected to an intein-C.
  • the polynucleotide encodes a PEgRNA.
  • the polypeptide encodes two or more components of a prime editing composition, for example, a prime editor fusion protein and a PEgRNA.
  • the polynucleotide encoding one or more prime editing composition components that is delivered to a target cell is integrated into the genome of the cell for long-term expression, for example, by a retroviral vector.
  • the polynucleotide delivered to a target cell is expressed transiently.
  • the polynucleotide may be delivered in the form of mRNA, or a non-integrating vector (non-integrating virus, plasmids, minicircle DNAs) for episomal expression.
  • a polynucleotide encoding one or more prime editing system components can be operably linked to a regulatory element, e.g., a transcriptional control element, such as a promoter.
  • a transcriptional control element such as a promoter.
  • the polynucleotide is operably linked to multiple control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc.
  • Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • the polynucleotide is provided as an RNA, e.g., a mRNA or a transcript.
  • RNA of the prime editing systems for example a guide RNA or a base editor- encoding mRNA, can be delivered in the form of RNA.
  • one or more components of the prime editing system that are RNAs is produced by direct chemical synthesis or may be transcribed in vitro from a DNA.
  • mRNA that encodes a prime editor polypeptide is generated using in vitro transcription.
  • Guide polynucleotides can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence “GG”, and guide polynucleotide sequence.
  • the prime editor encoding mRNA and/or PEgRNA(s) are synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA can directly contact a target TRAC gene or can be introduced into a cell using any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, or transfection).
  • the WSGR Docket No.59761-772601 prime editor-coding sequences and/or the PEgRNAs are modified to include one or more modified nucleoside e.g., using pseudo-U or 5-Methyl-C.
  • Methods of non-viral delivery of nucleic acids can include lipofection, electroporation, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, nanoparticles, cell penetrating peptides and associated conjugated molecules and chemistry, naked DNA, artificial virions, cell membrane disruption by a microfluidics device, and agent-enhanced uptake of DNA.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.
  • Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. RNA or DNA viral based systems can be used to target specific cells and trafficking the viral payload to an organelle of the cell. Viral vectors can be administered directly (in vivo) or they can be used to treat cells in vitro, and the modified cells can optionally be administered after delivery (ex vivo).
  • the viral vector is a retroviral, lentiviral, adenoviral, adeno-associated viral or herpes simplex viral vector.
  • Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector is a gamma retroviral vector.
  • the viral vector is an adenoviral vector.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • polynucleotides encoding one or more prime editing composition components are packaged in a virus particle.
  • Packaging cells can be used to form virus particles that can infect a target cell. Such cells can include 293 cells, (e.g., for packaging adenovirus), and ⁇ 2 cells or PA317 cells (e.g., for packaging retrovirus).
  • Viral vectors can be generated by producing a cell line that packages a nucleic acid vector into a viral particle.
  • the vectors can contain the minimal viral sequences required for packaging and subsequent integration into a host.
  • the vectors can contain other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed.
  • the missing viral functions can be supplied in trans by the packaging cell line.
  • AAV vectors can comprise ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • the polynucleotides are a DNA polynucleotide.
  • the polynucleotides are an RNA polynucleotide; e.g., an mRNA polynucleotide.
  • WSGR Docket No.59761-772601 [0719]
  • the AAV vector is selected for tropism to a particular cell, tissue, organism.
  • the AAV vector is pseudotyped, e.g., AAV5/8.
  • polynucleotides encoding one or more prime editing composition components are packaged in a first AAV and a second AAV. In some embodiments, the polynucleotides encoding one or more prime editing composition components are packaged in a first rAAV and a second rAAV.
  • dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5’ and 3’ ends that encode N-terminal portion and C- terminal portion of, e.g., a prime editor polypeptide), where each half of the cassette is no more than 5kb in length, optionally no more than 4.7 kb in length, and is packaged in a single AAV vector.
  • the full-length transgene expression cassette is reassembled upon co-infection of the same cell by both dual AAV vectors.
  • a portion or fragment of a prime editor polypeptide e.g., a Cas9 nickase
  • the portion or fragment of the polypeptide can be fused to the N-terminus or the C-terminus of the intein.
  • an N-terminal portion of the polypeptide is fused to an intein-N, and a C-terminal portion of the polypeptide is separately fused to an intein-C.
  • a portion or fragment of a prime editor fusion protein is fused to an intein and fused to an AAV capsid protein.
  • intein-N may be fused to the N-terminal portion of a first domain described herein
  • intein-C may be fused to the C-terminal portion of a second domain described herein for the joining of the N- terminal portion to the C-terminal portion, thereby joining the first and second domains.
  • the first and second domains are each independently chosen from a DNA binding domain or a DNA polymerase domain.
  • intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.).
  • a polynucleotide encoding a prime editor fusion protein is split in two separate halves, each encoding a portion of the prime editor fusion protein and separately fused to an intein.
  • each of the two halves of the polynucleotide is packaged in an individual AAV vector of a dual AAV vector system.
  • each of the two halves of the polynucleotide is no more than 5kb in length, optionally no more than 4.7 kb in length.
  • the full-length prime editor fusion protein is reassembled upon co-infection of the same cell by both dual AAV vectors expression of both halves of the prime editor fusion protein, and self- excision of the inteins.
  • the in vivo use of dual AAV vectors results in the expression of full-length prime editor fusion proteins.
  • the use of the dual AAV vector platform allows viable delivery of transgenes of greater than about 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size.
  • an intein is inserted at a splice site within a Cas protein. In some embodiments, insertion of an intein disrupts a Cas activity.
  • intein refers to a self- splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins WSGR Docket No.59761-772601 (e.g., fragments to be joined).
  • an intein may comprise a polypeptide that is able to excise itself and join exteins with a peptide bond (e.g., protein splicing).
  • an intein of a precursor gene comes from two genes (e.g., split intein).
  • an intein may be a synthetic intein.
  • Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: dnaE-n and dnaE-c. a 4-hydroxytamoxifen (4-HT)-responsive intein, an iCas molecule, a Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, Cfa DnaE intein, Ssp GyrB intein, and Rma DnaB intein.
  • intein fragments may be fused to the N terminal and C-terminal portion of a split Cas protein respectively for joining the fragments of split Cas9.
  • the split Cas9 system may be used in general to bypass the packing limit of the viral delivery vehicles.
  • a split Cas9 may be a Type II CRISPR system Cas9.
  • a first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
  • the first portion of the Cas9 protein is the N-terminal fragment of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal fragment of the Cas9 protein.
  • a split site may be selected which are surface exposed due to the sterical need for protein splicing.
  • a Cas protein may be split into two fragments at any C, T, A, or S.
  • a Cas9 may be intein split at residues 203-204, 280-292, 292-364, 311-325, 417- 438, 445-483, 468-469, 481-502, 513-520, 522-530, 565-637, 696-707, 713-714, 795-804, 803-810, 878-887, and 1153-1154.
  • protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574.
  • split Cas9 fragments across different split pairs yield combinations that provided the complete polypeptide sequence activate gene expression even when fragments are partially redundant.
  • a functional Cas9 protein may be reconstituted from two inactive split-Cas9 peptides in the presence of gRNA by using a split-intein protein splicing strategy.
  • the split Cas9 fragments are fused to either a N- terminal intein fragment or a C-terminal intein fragment, which can associate with each other and catalytically splice the two split Cas9 fragments into a functional reconstituted Cas9 protein.
  • a split-Cas9 can be packaged into self-complementary AAV.
  • a split-Cas9 comprises a 2.5 kb and a 2.2 kb fragment of S. pyogenes Cas9 coding sequences.
  • a split-Cas9 architecture reduces the length and/or size of the coding sequences of a viral vector, e.g., AAV.
  • a target cell can be transiently or non-transiently transfected with one or more vectors described herein.
  • a cell can be transfected as it naturally occurs in a subject.
  • a cell can be taken or WSGR Docket No.59761-772601 derived from a subject and transfected.
  • a cell can be derived from cells taken from a subject, such as a cell line.
  • a cell transfected with one or more vectors described herein can be used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the compositions of the disclosure (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editor, can be used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • Any suitable vector compatible with the host cell can be used with the methods of the disclosure.
  • Non-limiting examples of vectors include pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40.
  • a prime editor protein can be provided to cells as a polypeptide.
  • the prime editor protein is fused to a polypeptide domain that increases solubility of the protein.
  • the prime editor protein is formulated to improve solubility of the protein.
  • a prime editor polypeptide is fused to a polypeptide permeant domain to promote uptake by the cell.
  • the permeant domain is a peptide, a peptidomimetic, or a non-peptide carrier.
  • a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 1248).
  • the permeant peptide can comprise the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein.
  • Other permeant domains can include poly-arginine motifs, for example, the region of amino acids 34- 56 of HIV-1 rev protein, nona-arginine (SEQ ID NO: 1249), and octa-arginine (SEQ ID NO: 1250).
  • the nona-arginine (R9) sequence can be used.
  • the site at which the fusion can be made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide.
  • a prime editor polypeptide is produced in vitro or by host cells, and it may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc. and may be further refolded.
  • a prime editor polypeptide is prepared by in vitro synthesis.
  • Various commercial synthetic apparatuses can be used. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids.
  • a prime editor polypeptide is isolated and purified in accordance with recombinant synthesis methods, for example, by expression in a host cell and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • a prime editing composition for example, prime editor polypeptide components and PEgRNA(s) are introduced to a target cell by nanoparticles.
  • the prime editor polypeptide components and the PEgRNA form a complex in the nanoparticle. Any WSGR Docket No.59761-772601 suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components.
  • the nanoparticle is inorganic. In some embodiments, the nanoparticle is organic. In some embodiments, a prime editing composition is delivered to a target cell, e.g., a muscle cell, in an organic nanoparticle, e.g., a lipid nanoparticle (LNP) or polymer nanoparticle.
  • LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof. In some embodiments, neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability. In some embodiments, LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof.
  • Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 28 below. [0731]
  • components of a prime editing composition form a complex prior to delivery to a target cell. For example, a prime editor fusion protein and a PEgRNA can form a complex prior to delivery to the target cell.
  • a prime editing polypeptide e.g., a prime editor fusion protein
  • a guide polynucleotide e.g., a PEgRNA
  • the RNP comprises a prime editor fusion protein in complex with a PEgRNA.
  • RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, or any other approaches known in the art.
  • delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell.
  • the RNP comprising the prime editing complex is degraded over time in the target cell.
  • Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 28 below. Table 28. Exemplary lipids for nanoparticle formulation or gene transfer Lipid Abbreviation Feature 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine DOPC Helper 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE Helper Cholesterol Helper N41-(2,3-Dioleyloxy)prophyliN,N,N-trimethylammonium DOTMA Cationic chloride 1,2-Dioleoyloxy-3-trimethylammonium-propane DOGS Cationic Dioctadecylamidoglycylspermine N-(3-Aminopropy1)-N,N-dimethy1-2,3-bis(dodecyloxy)-
  • compositions may be provided to the subject cells one or more times, e.g., one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g., 16-24 hours.
  • the compositions may be delivered simultaneously (e.g., as two polypeptides and/or nucleic acids).
  • the prime editing compositions and pharmaceutical compositions of the disclosure can be administered to subjects in need thereof for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days.
  • compositions may be provided to the subject one or more times, e.g., one time, twice, three times, or more than three times.
  • different prime editing system components e.g., two different polynucleotide constructs are administered to the subject (e.g., different components of the same prime editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes)
  • the compositions may be administered simultaneously (e.g., as two polypeptides and/or nucleic acids).
  • they may be provided sequentially, e.g., one composition being provided first, followed by a second composition.
  • compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, and/or prime editing complexes described herein.
  • pharmaceutical compositions comprising the edited cells, e.g., T cells, described herein.
  • the pharmaceutical compositions of the present disclosure can be used to treat a disease, disorder, or a condition.
  • pharmaceutical composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
  • a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, and physiologic pH).
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • WSGR Docket No.59761-772601 EXAMPLES [0740] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.
  • PEgRNA libraries may be assembled by one of three cloning methods: in the first method, pooled synthesized DNA oligos encoding the PEgRNA and flanking U6 expression plasmid homology regions may be cloned into U6 expression plasmids via Gibson cloning and sequencing of bacterial colonies via Sanger or Next-generation sequencing. In the second method, double-stranded linear DNA fragments encoding PEgRNA and homology sequences as above may be individually Gibson-cloned into U6 expression plasmids.
  • oligos encoding a protospacer, a gRNA scaffold, and PEgRNA extension may be ligated, and then cloned into a U6 expression plasmid as described in Anzalone et al., Nature. 2019 Dec; 576(7785):149-157. Bacterial colonies carrying sequence-verified plasmids may then be propagated in LB or TB. Plasmid DNA may be purified by minipreps for mammalian transfection. Alternatively, PEgRNAs may be chemically synthesized and modified.
  • HEK293T cells may be propagated in DMEM with 10% FBS. For 96 well-plates, the cells may be seeded at 10,000 cells per well 24 hours prior to transfection. For 48-well plates, the cells may be seeded at 30,000 cells per well 24 hours prior to transfection. Seeded cells may be transfected with Lipofectamine 2000 according to the manufacturer’s directions with PEgRNAs and plasmid DNA encoding a prime editor fusion protein. Three days after transfection, genomic DNA may be harvested in lysis buffer for high throughput sequencing (e.g., using Miseq).
  • T cells may be cultured in OpTimizer CTS T Cell Expansion SFM containing 5% CTS Immune Cell SR (ThermoFisher), L-Glutamine, Penicillin/Streptomycin (Lonza), 10 mM N-Acetyl-L-cysteine (Sigma-Aldrich, St. Louis, MO), 300 IU/mL IL-2, 5 ng/mL IL7, and 5 ng/mL IL-15 (PeproTech).
  • T cells are activated with DynaBeads Human T-Activator CD3/CD28 (ThermoFisher) at a 2:1 bead:cell ratio for 48 hours prior to electroporation.
  • DynaBeads may be magnetically removed, and T cells washed once with PBS prior to resuspension in electroporation buffer.
  • mRNA encoding a prime editor fusion protein and chemically synthesized pegRNAs are suspended in electroporation buffer and mixed with T cells.
  • mRNA encoding the recombinase (Bxb1) and the DNA donor may be similarly suspended in electroporation buffer and mixed and with T cells. Cells may be electroporated according to the manufacturer’s protocol (MaxCyte).
  • T cells may be transferred to an empty well-plate and allowed to recover at 37 °C, 5% CO 2 for 20 minutes. Subsequently, cells are suspended in basal medium (without serum replacement or cytokines) and incubated at 37 °C, 5% WSGR Docket No.59761-772601 CO 2 for 20 minutes before being suspended in complete T cell medium. Two days after electroporation, T cells may be transferred to G-Rex rapid expansion culture vessels. Genomic DNA may be extracted using Lucigen QuickExtraction Solution according to manufacturer’s protocol for sequence analysis.
  • EXAMPLE 2 Dual prime editing of the TRAC gene in HEK 293T cells
  • PEgRNA pairs were designed for a dual prime editing strategy such that the protospacers of a PEgRNA pair flank the exon 1/intron 1 junction of the TRAC gene.
  • the design would allow introduction of a recombinase recognition site at such genomic locations that a potential transgene (e.g. a transgene on a donor DNA) sequence integrated at the locations can be expressed using either an exogenous promoter (e.g., as part of the transgene) or the endogenous TRAC promoter.
  • 4 spacers were used with varying PBS lengths from 8 to 13nt.
  • the 5’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 27, and the 3’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 107 (full complementarity between the 5’ RTT and 3’ RTT, each encoding the 38bp attB sequence/reverse complement thereof). All of the PEgRNAs contained a gRNA core sequence according to SEQ ID NO: 590.
  • PEgRNAs designed as above were assembled from U6 expression plasmids, and HEK293T cells were transfected with PEgRNA pairs and plasmid encoding a prime editor fusion protein as described in Example 1.72 hours after transfection, genomic DNA was extracted by aspirating media, adding QuickExtract DNA Extraction solution (Lucigen, #QR9050) and incubating according to manufacturer’s protocol for high-throughput sequencing.467 PEgRNA pairs were tested to examine each 5’ PEgRNA spacer paired with each 3’ PEgRNA spacer having varying PBS lengths. Sequence numbers of the PEgRNAs and PEgRNA components, as well as editing efficiency and indels are summarized in Tables 31A-31D.
  • the PEgRNAs in Tablesx1A-D will contain, from 5’ to 3’, the indicated PEgRNA sequence and a variable number of 3’ Us (e.g., up to 4 Us).
  • the editing efficiency and indel frequency scales in Tables 31A-31D are as follows. Editing efficiency: 0.3%-15%: +; 15%-29%: ++; 29%-43%: +++; 43%-58%: ++++.
  • Table 31A Dual prime editing efficiency in HEK293T cells.
  • All 5’ PEgRNAs have a spacer sequence according to SEQ ID NO: 4.
  • the 5’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 27.
  • the 3’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 107.
  • All 5’ PEgRNAs have a spacer sequence according to SEQ ID NO: 61.
  • the 5’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 27.
  • the 3’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 107.
  • All 5’ PEgRNAs have a spacer sequence according to SEQ ID NO: 88.
  • the 5’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 27.
  • the 3’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 107.
  • All 5’ PEgRNAs have a spacer sequence according to SEQ ID NO: 150.
  • the 5’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 27.
  • the 3’ PEgRNAs contain an RTT sequence according to SEQ ID NO: 107.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
EP24739021.4A 2023-01-05 2024-01-05 Genomeditierungszusammensetzungen und verfahren zur verwendung Pending EP4646482A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202363478654P 2023-01-05 2023-01-05
US202363603479P 2023-11-28 2023-11-28
PCT/US2024/010561 WO2024148313A2 (en) 2023-01-05 2024-01-05 Genome editing compositions and methods of use

Publications (1)

Publication Number Publication Date
EP4646482A2 true EP4646482A2 (de) 2025-11-12

Family

ID=91804406

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24739021.4A Pending EP4646482A2 (de) 2023-01-05 2024-01-05 Genomeditierungszusammensetzungen und verfahren zur verwendung

Country Status (8)

Country Link
EP (1) EP4646482A2 (de)
JP (1) JP2026503266A (de)
KR (1) KR20250155074A (de)
CN (1) CN121586577A (de)
AU (1) AU2024206326A1 (de)
IL (1) IL321929A (de)
MX (1) MX2025007891A (de)
WO (1) WO2024148313A2 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4703469A1 (de) * 2024-08-26 2026-03-04 Charité - Universitätsmedizin Berlin Körperschaft des öffentlichen Rechts Fusionsproteine, verfahren und zusammensetzungen für effizienten serinintegrasevermittelten gentransfer in menschlichen zellen
WO2026046974A1 (en) * 2024-08-26 2026-03-05 Charité - Universitätsmedizin Berlin Körperschaft des öffentlichen Rechts Fusion proteins, methods and compositions for efficient serine integrase-mediated gene transfer in human cells

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL297761A (en) * 2020-05-08 2022-12-01 Broad Inst Inc Methods and compositions for simultaneously editing two helices of a designated double-helix nucleotide sequence
EP4232583A1 (de) * 2020-10-21 2023-08-30 Massachusetts Institute of Technology Systeme, verfahren und zusammensetzungen für ortsspezifisches gen-engineering unter verwendung programmierbarer addition über ortsspezifische targeting-elemente (paste)

Also Published As

Publication number Publication date
IL321929A (en) 2025-09-01
WO2024148313A2 (en) 2024-07-11
AU2024206326A1 (en) 2025-07-31
JP2026503266A (ja) 2026-01-28
WO2024148313A3 (en) 2024-09-19
CN121586577A (zh) 2026-02-27
MX2025007891A (es) 2025-10-01
KR20250155074A (ko) 2025-10-29

Similar Documents

Publication Publication Date Title
US20240067940A1 (en) Methods and compositions for editing nucleotide sequences
US20250243474A1 (en) Genome editing compositions and methods for treatment of chronic granulomatous disease
US20240424138A1 (en) Genome editing compositions and method for treatment of retinitis pigmentosa
US20240382620A1 (en) Genome editing compositions and methods for treatment of usher syndrome type 3
US20240229038A1 (en) Genome editing compositions and methods for treatment of wilson's disease
CA3221154A1 (en) Genome editing compositions and methods for treatment of wilson's disease
US20240360476A1 (en) Genome Editing Compositions and Methods for Treatment of Myotonic Dystrophy
US20250073352A1 (en) Genome editing compositions and methods for treatment of fragile x syndrome
US20250319202A1 (en) Genome editing compositions and methods for treatment of fuchs endothelial corneal dystrophy
US20250011766A1 (en) Genome Editing Compositions and Methods for Treatment of Amyotrophic Lateral Sclerosis
AU2024206326A1 (en) Genome editing compositions and methods of use
US20240352453A1 (en) Genome editing compositions and methods for treatment of retinopathy
US20250376674A1 (en) Split prime editors
US20240376466A1 (en) Genome editing compositions and methods for treatment of fanconi anemia
WO2024163680A2 (en) Genome editing compositions and methods for treatment of cystic fibrosis
US20250179483A1 (en) Genome editing compositions and methods for treatment of glycogen storage disease type 1b
IL324508A (en) Genome editing compositions targeting the b2m gene and methods of use
WO2025090637A2 (en) Genome editing compositions and methods for treatment of retinitis pigmentosa
IL322419A (en) Genome editing agents and methods for treating cystic fibrosis

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250723

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)