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WO2024178144A1 - Procédés et compositions pour la réécriture de séquences nucléotidiques - Google Patents

Procédés et compositions pour la réécriture de séquences nucléotidiques Download PDF

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WO2024178144A1
WO2024178144A1 PCT/US2024/016758 US2024016758W WO2024178144A1 WO 2024178144 A1 WO2024178144 A1 WO 2024178144A1 US 2024016758 W US2024016758 W US 2024016758W WO 2024178144 A1 WO2024178144 A1 WO 2024178144A1
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domain
sequence
dna
amino acid
fold
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Christopher Wilson
Andrew V. Anzalone
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Prime Medicine Inc
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Prime Medicine Inc
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • An effective genome editing technique also needs to be customable; modulable; and programmable, suitable of making any genome changes in any cells or organisms. Furthermore, an effective genome editing technique needs to scalable and reliable, proficient in making any genome changes reproducibly in a robust scale. INCORPORATION BY REFERENCE [0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.
  • the present disclosure provides a prime editing composition that comprises a) a DNA binding domain or one or more polynucleotides encoding the DNA binding domain; and b) a DNA polymerase domain (e.g., a reverse transcriptase or an engineered reverse transcriptase) or one or more polynucleotides encoding the DNA polymerase domain, wherein the DNA binding domain comprises an amino acid sequence of a Cas12i protein, e.g., a Cas 12i1 protein, a Cas12i2 protein, a Cas12i3 protein or a Cas12i4 protein, or a domain of a Cas12i protein, and the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-80, 81-95, 129-136, 198-271, 319- 493, 533-846, 855-8
  • the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 81-95, 129-136, 204, 209, 210, 229- 244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 345, 396, 489, 533-846, 855-857, 884, 990-1005 and 1006.
  • the amino acid sequence of the DNA polymerase domain has at least about 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, WSGR Docket No.59761-781.601 97%, 98%, 99%, or 100% sequence identity to the selected sequence.
  • the selected sequence for the DNA polymerase domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, 81, 82, 85, 91, 209, 210, 261, 270, 855, 856, 857 and 884.
  • the present disclosure provides a prime editing composition that comprises: a) a DNA binding domain comprising a Cas12i protein or domain thereof, or one or more polynucleotides encoding the DNA binding domain; and b) a DNA polymerase domain or one or more polynucleotides encoding the DNA polymerase domain, wherein the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to a sequence selected from SEQ ID NOs: 1048-1215, or 1217-1395.
  • the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to any one of sequences set forth in SEQ ID NOs: 1177.
  • the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to any one of sequences set forth in SEQ ID NOs: 1267, 1288-1291. [0008] In some embodiments, the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to any one of sequences set forth in SEQ ID NOs: 1308, 1312, 1315, 1316, 1325, or 1326. [0009] In some embodiments, the DNA polymerase domain comprises an amino acid sequence with at least 85% sequence identity to any one of sequences set forth in SEQ ID NOs: 1105 or 1367.
  • the amino acid sequence of the DNA polymerase domain has at least about 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the selected sequence.
  • the DNA binding domain comprises a Cas12i1 protein or domain thereof.
  • the DNA binding domain comprises a Cas12i2 protein or domain thereof.
  • the Cas12i2 protein or domain thereof is a nickase.
  • the Cas12i2 protein or domain thereof is an endonuclease.
  • the Cas12i2 protein or domain thereof comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 1017. In some embodiments, the Cas12i2 protein or domain thereof comprises an amino acid sequence with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1017. In some embodiments, the Cas12i2 protein or domain thereof comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1017.
  • the Cas12i2 protein or domain thereof comprises an amino acid sequence comprising one or more mutations at positions D581, G624, F626, P868, I926R, V1030, E1035, and/or S1046 relative to SEQ ID NO: 1017.
  • the Cas12i2 protein or domain thereof comprises an amino acid sequence comprising one or more amino acid substitutions selected from D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G relative to SEQ ID NO: 1017.
  • the Cas12i2 protein or domain thereof comprises an amino acid sequence selected from SEQ ID NOs: 1018, 1019, 1020, 1021 or 1022. In some embodiments, the protein or domain thereof comprises an amino acid sequence selected from SEQ ID NOs: 1019 or 1022.
  • the DNA binding domain comprises a Cas12i protein or domain thereof that is a Cas12i1 protein or domain thereof, a Cas12i3 protein or domain thereof, or a Cas12i4 protein or a domain thereof. In some embodiments, the Cas12i protein or domain thereof is a nickase.
  • the Cas12i protein or domain thereof is an endonuclease.
  • the Cas12i protein or domain thereof comprises an amino acid sequence with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 1023, 1024, 1025 or 1026.
  • the amino acid sequence of the DNA binding domain has at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1023, 1024, 1025 and 1026.
  • the Cas12i2 protein or domain thereof comprises an amino acid sequence selected from SEQ ID NOs: 1023, 1024, 1025 or 1026.
  • the DNA binding domain is connected to the DNA polymerase domain by a linker.
  • the DNA binding domain is connected to the DNA polymerase domain by a peptide linker in a fusion protein.
  • the peptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 272-318, 913-926 and 1014.
  • the fusion protein comprises the DNA polymerase domain and the DNA binding domain from N-terminus to C-Terminus.
  • the fusion protein comprises the DNA binding domain and the DNA polymerase domain from N-terminus to C-Terminus.
  • the DNA polymerase domain, the DNA binding domain, or both comprise one or more nuclear localization signals.
  • the primer editing composition further comprises a solubility-enhancement (SET) domain.
  • the SET domain comprises an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-124 and 137.
  • the amino acid sequence for the SET domain has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the selected sequence.
  • the selected sequence for the SET domain is SEQ ID NO: 102 or 137.
  • the SET domain is fused to the DNA polymerase via an SGGS linker.
  • the primer editing composition further comprises a nuclear localization signal (NLS).
  • the NLS comprises an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 522-532, 928-931, 1015 and 1016.
  • the amino acid sequence for the NLS has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the selected sequence.
  • the prime editing composition of any one of the embodiments herein further comprises a recombinase protein domain or a polynucleotide encoding the recombinase protein domain.
  • the recombinase protein domain is selected form a recombinase of Tables 11, 12, 13, or 14.
  • the present disclosure provides a prime editing composition that comprises the DNA binding domain, or a polynucleotide encoding the DNA binding domain, and the DNA polymerase domain or a polynucleotide encoding the DNA polymerase domain, wherein the DNA binding domain and the DNA polymerase domain are on separate polypeptides.
  • the present disclosure provides a fusion prime editing protein or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises the DNA binding domain connected to the DNA polymerization domain.
  • the fusion protein comprises the DNA binding domain is connected to the DNA polymerization domain via a linker.
  • the DNA binding domain is connected to the DNA polymerase domain by a peptide linker in a fusion protein.
  • the peptide linker comprises an amino acid sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 272-318, 913-926 and 1014.
  • the amino acid sequence of the peptide linker has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the selected sequence.
  • the fusion protein comprises the DNA polymerase domain and the DNA binding domain from N-terminus to C-Terminus. In some embodiments, the fusion protein comprises the DNA binding domain and the DNA polymerase domain from N-terminus to C- Terminus. In some embodiments, the fusion protein comprises a nuclear localization signal, the DNA binding domain, the peptide linker, the DNA polymerase domain, the SGGS linker, the SET domain, and a second nuclear localization signal from N-terminus to C-terminus. In some embodiments, the present disclosure provides a fusion prime editing protein or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises an additional protein domain.
  • the present disclosure provides a fusion prime editing protein or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises a recombinase protein domain.
  • the sequence identity of a fusion protein, a DNA binding domain, a DNA polymerase domain, and/or a peptide linker 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 to the reference polypeptide.
  • the prime editing composition further comprises a prime editing guide RNA (PEgRNA), or a polynucleotide encoding the PEgRNA.
  • the prime editing composition further comprises a nick guide RNA (ngRNA), or a polynucleotide encoding the ngRNA.
  • PEgRNA prime editing guide RNA
  • ngRNA nick guide RNA
  • the PEgRNA comprises a DNA synthesis template comprising a recombinase recognition sequence recognized by the recombinase protein domain
  • the recombinase recognition sequence is selected from the recombinase recognition sequences in Tables 11-14 and wherein the recombinase protein domain comprises a corresponding recombinase in the same row of the selected recombinase recognition sequence
  • the prime editing composition further comprises a donor sequence or a polynucleotide encoding the donor sequence wherein the donor sequence comprises a second recombinase recognition sequence recognized by the recombinase protein domain.
  • the prime editing composition of any one of the embodiments disclosed herein further comprises: (i) a first prime editing guide RNA (PEgRNA) or a polynucleotide encoding the first PEgRNA, wherein the first PEgRNA comprises a first spacer, a first gRNA core, and a first DNA synthesis template, wherein the first spacer binds to a first sequence in the target DNA, and (ii) a second prime editing guide RNA (PEgRNA) or a polynucleotide encoding the second PEgRNA, wherein the second PEgRNA comprises a second spacer, a second gRNA core, and a second DNA synthesis template, wherein the second spacer binds to a second sequence in the target DNA; wherein the first spacer is complementary to a first strand of a double stranded target DNA, wherein the second spacer is complementary to a second strand of the double stranded target DNA complementary to the first strand, and where
  • PEgRNA prime editing guide
  • the first DNA synthesis template comprises a region of complementarity to the second DNA synthesis template, optionally wherein at least 10nt at the 5’ ends of the first and second DNA synthesis templates have perfect reverse complementarity to each other, optionally wherein the first and second DNA synthesis templates are fully complementary to each other.
  • the first DNA synthesis template comprises a recombinase recognition sequence or a fragment thereof.
  • the first DNA synthesis template comprises a recombinase recognition sequence selected from recombinase recognition sequences in Tables 11-14 or a 5’ fragment of the selected sequence, and wherein the second DNA synthesis template comprises a reverse complement of the selected sequence or a 5’ fragment of the reverse complement.
  • the first DNA synthesis template comprises in full length the selected recombinase recognition sequence.
  • the second DNA synthesis template comprises in full length the reverse complement of the selected recombinase recognition sequence.
  • the prime editing composition of any one of the embodiments further comprises a recombinase protein domain or a polynucleotide encoding the recombinase protein WSGR Docket No.59761-781.601 domain, wherein the recombinase protein domain is capable of recognizing the recombinase recognition sequence.
  • the prime editing composition of any one of the embodiments further comprises a donor sequence or a polynucleotide encoding the donor sequence, wherein the donor sequence comprises a second recombinase recognition sequence recognized by the recombinase protein domain.
  • the first DNA synthesis template comprises a recombinase recognition sequence selected from recombinase recognition sequences #1 of Tables 11, 12 or 14 or a 5’ fragment of the selected sequence, and wherein the donor sequence comprises a corresponding recombinase recognition sequence #2 in the same row as the selected recombinase recognition sequence #1; or (ii) the first DNA synthesis template comprises a recombinase recognition sequence selected from recombinase recognition sequences #2 of Tables 11, 12 or 14 or a 5’ fragment of the selected sequence, and wherein the donor sequence comprises a corresponding recombinase recognition sequence #1 in the same row as the selected recombinase recognition sequence #2.
  • the first DNA synthesis template comprises a recombinase recognition sequence selected from recombinase recognition sequences of Table 13 or a 5’ fragment of the selected sequence, and wherein the donor sequence comprises the selected sequence.
  • the selected recombinase recognition sequence is SEQ ID NO: 1427.
  • the selected recombinase recognition sequence is SEQ ID NO: 1396.
  • one or more of the polynucleotides of the prime editing composition is mRNA.
  • the present disclosure provides a vector comprising one or more of the polynucleotides of the prime editing compositions of the present disclosure.
  • the vector is a viral vector.
  • the vector is an AAV vector.
  • the vector is a non-viral vector.
  • the non-viral vector is a plasmid.
  • the present disclosure provides a lipid nanoparticle (LNP) comprising one or more of the polynucleotides of the prime editing compositions of the present disclosure.
  • the present disclosure provides a ribonucleoprotein (RNP) comprising one or more of the proteins of the prime editing compositions of the present disclosure.
  • LNP lipid nanoparticle
  • RNP ribonucleoprotein
  • the present disclosure provides a pharmaceutical composition comprising the prime editing composition, a vector, an LNP or an RNP of the present disclosure.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the present disclosure provides a method for editing a target DNA, comprising contacting the target DNA with a prime editing composition of any one of the WSGR Docket No.59761-781.601 embodiments disclosed herein, a vector of any one of the embodiments disclosed herein, an LNP disclosed herein, or a pharmaceutical composition of any one of the embodiments disclosed herein.
  • FIG.1 shows a cartoon illustration of the domain structure of an exemplary prime editor comprising a DNA binding domain (e.g., a Cas12i2 protein domain) and a DNA polymerase domain (e.g., a reverse transcriptase domain) connected by a linker.
  • FIG.2 shows illustrations of unstructured, structured, and natural linker variants useful in the prime editors disclosed herein.
  • FIG.3 contains schematics for six different RT families. The domain comprising conserved sequences are illustrated on the top. The specific amino acid and sequence motif at each domain for various families are also shown. Sequences of conserved motifs, e.g., SEQ ID NOs: 905-909 and 1101-1102, respectively, are indicated for each RT family in order of appearance.
  • FIG.4A contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA. Triangles indicate exemplary single stranded breaks generated by a prime editor containing a nuclease active Cas nuclease.
  • FIG.4B contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA.
  • Triangles indicate exemplary single stranded breaks generated by a prime editor containing a nuclease active Cas nuclease. In cases where the prime editor contains a Cas nickase, only one single stranded break indicated by one of the two triangles would be generated for each of the two PEgRNAs.
  • FIG.4C contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA.
  • Triangles indicate exemplary single stranded breaks generated by a prime editor containing a nuclease active Cas nuclease. In cases where the prime editor contains a Cas nickase, only one single stranded break indicated by one of the two triangles would be generated for each of the two PEgRNAs. Same color/shading indicates complementarity or identity between two sequences.
  • FIG.4D contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA.
  • FIG.4E contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between two sequences.
  • FIG.4F contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between two sequences.
  • FIG.4G contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between two sequences.
  • FIG.4H contains a schematic showing a pair PEgRNAs of an exemplary dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between two sequences.
  • DETAILED DESCRIPTION OF THE DISCLOSURE Provided herein, in some embodiments, are compositions and methods related to prime editors.
  • the prime editors (PEs) provided herein can 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 that serve a variety of functions, including correction of disease-causing mutations.
  • PEgRNAs prime editing guide RNAs
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • a “cell” can generally refer 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. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an animal cell, a cell from an invertebrate animal (e.g.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non- human primate, a human, etc.
  • 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 human cell can be of or derived from different tissues, organs, and/or cell types.
  • the human cell is a primary cell.
  • the term “primary cell” means a cell isolated from an organism, e.g., a human, 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
  • the cell is a human stem cell.
  • human 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.
  • prime editing compositions e.g., through transfection, transduction, electroporation, and the like
  • Such modified human cells include muscle cells (e.g., cardiac muscle cells, smooth muscle cells, hepatocytes), hematopoietic stem cells (HSCs), hematopoietic stem progenitor cells (HSPCs), fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.
  • the human cell is a human stem cell.
  • the human cell is a human progenitor cell.
  • the human cell is a pluripotent human cell (e.g., a pluripotent stem cell).
  • the human cell e.g., a stem cell
  • the human cell is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell.
  • the human cell is an induced pluripotent stem cell (iPSC).
  • the human cell is an embryonic stem cell (ESC).
  • the human cell is a CD34 + cell.
  • the human cell is a hematopoietic stem cell (HSC).
  • the human cell is a hematopoietic progenitor cell (HPC).
  • HPC hematopoietic progenitor cell
  • hematopoietic stem cells and hematopoietic progenitor cells are referred to as hematopoietic stem or progenitor cells (HSPCs).
  • the cell is a human HSC.
  • the cell is a human HPC.
  • the cell is a human HSPC.
  • the human cell is a long term (LT)-HSC.
  • ST short-term
  • the human cell is a myeloid progenitor cell.
  • the human cell is a lymphoid progenitor cell. In some embodiments, the human cell is a granulocyte monocyte progenitor cell. In some embodiments, the human cell is a megakaryocyte erythroid progenitor cell. In some embodiments, the human cell is a multipotent progenitor cell (MPP). [0064] In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human hematopoietic stem cell (HSC) or a human hematopoietic stem and progenitor cell. In some embodiments, the human HSC is from bone marrow or mobilized peripheral blood.
  • HSC human hematopoietic stem cell
  • the human HSC is from bone marrow or mobilized peripheral blood.
  • the human stem cell is an induced pluripotent stem cell (iPSC).
  • the cell is a human HSC.
  • the cell is a human CD34 + cell.
  • the human cell is a hematopoietic progenitor cell, multipotent progenitor cell, lymphoid progenitor cell, a myeloid progenitor cell, a megakaryocyte-erythroid progenitor cell, a granulocyte- megakaryocyte progenitor cell, a granulocyte, a promyelocyte, a neutrophil, an eosinophil, a basophil, an erythrocyte, a reticulocyte, a thrombocyte, a megakaryoblast, a platelet-producing megakaryocyte, a monocyte, a macrophage, a dendritic cell, a microglia, an osteoclast, a lymphocyte, a NK cell,
  • the human cell edited by prime editing can be differentiated into, or give rise to recovery of a population of human cells, e.g., common lymphoid progenitor cells, WSGR Docket No.59761-781.601 common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, granulocyte- megakaryocyte progenitor cells, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, such as NK cells, B-cells or T-cells.
  • human cells e.g., common lymphoid progenitor cells, WSGR Docket No.59761-781.601 common myeloid progenitor
  • the human cell edited by prime editing can be differentiated into, or give rise to recovery of a population of human cells, e.g., neutrophils, platelets, red blood cells, monocytes, macrophages, antigen-presenting cells, microglia, osteoclasts, dendritic cells, inner ear cell, inner ear support cell, cochlear cell and/or lymphocytes.
  • the human cell is in a subject, e.g., a human subject.
  • the cell is not isolated from an organism, e.g., a human, but forms part of a tissue or organ of the organism.
  • a cell is isolated from an organism, e.g., the human.
  • a cell is derived from an organism, e.g., a human.
  • a cell is a differentiated cell.
  • the cell is a fibroblast.
  • the cell is differentiated from an induced pluripotent stem cell.
  • the cell is differentiated from an HSC or an HPSC.
  • the cell is differentiated from an induced pluripotent stem cell (iPSC).
  • the cell is differentiated from an embryonic stem cell (ESC).
  • the cell is a differentiated human cell.
  • cell is a human fibroblast.
  • the cell is differentiated from an induced human pluripotent stem cell. In some embodiments, the cell is differentiated from a human iPSC or a human ESC. [0067] In some embodiments, the cell comprises a prime editor or a prime editing composition disclosed herein. In some embodiments, the cell comprises a prime editor, a PEgRNA, or a prime editing composition disclosed herein. In some embodiments, the cell is from a human subject. In some embodiments, the human subject has a disease or condition, or is at a risk of developing a disease or a condition associated with a mutation to be corrected by prime editing. In some embodiments, the cell comprises a mutation associated with a disease or disorder.
  • the cell is from a human subject, and comprises a prime editor, a PEgRNA, or a prime editing composition disclosed herein for correction of the mutation.
  • the cell is from the human subject, and the mutation has been edited or corrected by prime editing.
  • the cell is in a human subject.
  • the cell comprises a prime editor or a prime editing composition for correction of the mutation.
  • the cell is in a human subject, and comprises a prime editor or a prime editing composition for correction of the mutation.
  • the mutation in the cell has been edited or corrected by prime editing.
  • the cell is from the human subject and the mutation has been edited or corrected by prime editing.
  • the term can refer to an amount that can 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 can refer to an amount that can 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 can be a full-length protein (e.g., a fully processed protein having certain biological function).
  • a protein can be a variant or a fragment of a full-length 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 or portion thereof 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 comprises multiple protein domains from different naturally occurring proteins.
  • a prime editor can be a fusion protein comprising a Cas12i2 protein and a reverse transcriptase protein domain of a retrovirus (e.g., Moloney murine leukemia virus) or a variant of the retrovirus.
  • a retrovirus e.g., Moloney murine leukemia virus
  • a protein that comprises amino acid sequences from different origins or naturally occurring proteins can be referred to as a fusion, or chimeric protein.
  • 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., WSGR Docket No.59761-781.601 catalytic or binding functions.
  • a functional fragment of a reverse transcriptase can 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 can retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas12i2 can encompass less than the entire amino acid sequence of a Cas12i2 protein, but retains its DNA binding ability and lacks its nuclease activity partially or completely.
  • a functional fragment of a Cas12i2 nickase encompasses less than the entire amino acid sequence of the corresponding full-length protein but retains its DNA binding ability and has DNA nickase activity.
  • 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 can 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 can retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas12i2 can comprise one or more amino acid substitutions in a nuclease domain compared to the amino acid sequence of a wild type Cas12i2, but retains the DNA binding ability and lacks the nuclease activity partially or completely.
  • a functional fragment of a Cas12i2 can comprise one or more amino acid substitutions in a nuclease domain compared to the amino acid sequence of a wild type Cas12i2, but retains the DNA binding ability and has DNA nickase activity.
  • the term “function” and its grammatical equivalents as used herein refer to a capability of operating, having, or serving an intended purpose. Functional can comprise any percent from baseline to 100% of an intended purpose. For example, functional can 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.
  • a protein or polypeptides 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.
  • a “homologous sequence” of nucleic acid sequences can 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, a primer binding site, or a protospacer sequence to the 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 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.
  • WSGR Docket No.59761-781.601 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.
  • amino acid (or nucleotide) positions can be determined in homologous sequences based on alignment, for example, “R20” in a reference protein sequence may correspond to R19 in a protein sequence lacking an N-terminal methionine, or may correspond to another position in a homolog of such protein.
  • the term “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.
  • a polynucleotide is single-stranded or substantially single-stranded, WSGR Docket No.59761-781.601 e.g., single-stranded DNA or an mRNA.
  • a polynucleotide is a cell-free nucleic acid molecule.
  • a polynucleotide circulates in blood.
  • a polynucleotide is a cellular nucleic acid molecule.
  • a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • Polynucleotides can have any three-dimensional structure. The following are nonlimiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA
  • 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 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 a 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 WSGR Docket No.59761-781.601 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.
  • 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 of two polynucleotide molecules.
  • the portion 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, translated into peptides, polypeptides, or proteins.
  • expression may include splicing of the mRNA in a eukaryotic cell.
  • expression of a polynucleotide e.g., a gene or a DNA encoding a protein
  • expression of a polynucleotide 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.
  • expression of a gene is determined by the amount of the mRNA, or transcript, that is encoded by the gene after transcription the gene.
  • expression of a polynucleotide e.g., an mRNA
  • expression of a polynucleotide is determined by the amount of the protein encoded by the mRNA after translation of the mRNA.
  • expression of a polynucleotide e.g., a mRNA or coding RNA, 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.
  • WSGR Docket No.59761-781.601 The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, or biological or cellular material, and means a molecule having minimal homology to another molecule while still maintaining a desired structure or functionality.
  • 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 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.
  • the term “mutation” as used herein 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 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.
  • subject and its grammatical equivalents as used herein may refer to a human or a non-human organism.
  • 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 or fetus.
  • a human subject may be a newborn, an infant, a child, an adolescent, or an adult.
  • a human subject may be up to about 100 years of age.
  • a human subject may be in need of treatment for a genetic disease or disorder.
  • treatment 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 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.
  • effective amount or “therapeutically 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 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 (e.g., expression of a gene to produce functional a protein) 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, 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 (e.g., expression of a target gene to produce a functional protein).
  • target gene modulation e.g., expression of a target gene to produce a functional protein.
  • 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. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). [0099] An effective amount can be the amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have a correction a mutation.
  • an effective amount can be the amount to induce, when administered to or introduced WSGR Docket No.59761-781.601 to a population of cells, installation of one or more intended nucleotide edits that correct a mutation in the target gene, in at least about 1%, 2%, 5%, 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 about 99% of the population of cells.
  • DNA polymerase refers to a class of enzymes that synthesize a DNA molecule from a nucleic acid molecule template, e.g., a DNA template or an RNA template.
  • reverse transcriptase or “RT” as used herein refers to a class of DNA polymerases that synthesize a DNA molecule from an RNA template.
  • An RT may require the primer molecule with an exposed 3’ hydroxyl group.
  • the primer molecule of an RT may be a DNA molecule.
  • the primer molecule of an RT may be an RNA molecule.
  • an RT may comprise both DNA polymerase activity and RNase H activity.
  • linker refers to a bond, a chemical group, or a molecule linking two molecules or moieties, e.g., two p domains to form a fusion protein.
  • a linker can be a peptide linker.
  • a linker can also be a polynucleotide or oligonucleotide linker.
  • RNA-binding protein recruitment sequence such as a MS2 polynucleotide sequence
  • a RNA-binding protein recruitment sequence can be used to connect a Cas12i2 domain and a DNA polymerase domain of a prime editor, wherein one of the Cas12i2 domain and the DNA polymerase domain is fused to a MS2 coat protein.
  • a peptide linker may have various lengths, depending on the application of a linker or the sequences or molecules being linked by a linker.
  • SET domain refers to a group of protein or peptide domains that enhance the solubility of a second protein or polypeptide when expressed as a fusion protein or polypeptide, relative to the second protein or polypeptide when expressed alone.
  • a SET domain may also increase the activity of the second protein or polypeptide (e.g., enzymatic activity or nucleic acid- / protein-binding activity) when expressed as a fusion protein or polypeptide, relative to the second protein or polypeptide when expressed alone.
  • a SET domain may also increase the expression level of the second protein or polypeptide when expressed as a fusion protein or polypeptide, relative to the second protein or polypeptide when expressed alone.
  • a SET domain may also increase degree of folding to a native fold of the second protein or polypeptide when expressed as a fusion protein or polypeptide, relative to the second protein or polypeptide when expressed alone.
  • the term “nuclear localization signal” or “NLS” as used herein refers to a amino acid sequence that, when attached to a protein or polypeptide, targets the importation of the protein into the nucleus of a cell. An NLS, when attached to a protein, increases the amount of the protein imported into the cell nucleus compared to the protein not having an attached NLS.
  • fusion protein refers to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a WSGR Docket No.59761-781.601 different function.
  • a domain may comprise a particular makeup of amino acids.
  • a domain may also comprise a structure of proteins as described herein.
  • Prime Editing refers to programmable editing of a target DNA using a prime editor complexed with a prime editing guide RNA (PEgRNA) to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target DNA through target-primed DNA synthesis.
  • PEgRNA prime editing guide RNA
  • a target gene of prime editing can comprise a double stranded DNA molecule having two complementary strands: a first strand that can be referred to as a “target strand”, and a second strand that can be referred to as a “non-target strand”.
  • a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which can be referred to as a “search target sequence”.
  • search target sequence a specific sequence on the target strand
  • the spacer sequence anneals with the target strand at the search target sequence.
  • the target strand can 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 can have a substantially identical sequence as the protospacer sequence on the PAM strand of a target gene, except that the spacer sequence can comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).
  • U Uracil
  • T Thymine
  • Prime editing can be initiated on either the PAM strand or the non-PAM strand.
  • the double stranded target DNA comprises a nick site on the non-PAM strand. In some embodiments, 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. In some embodiments, 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 or a cleavage will occur when the double stranded target DNA is contacted with a nickase or a nuclease, for example, a Cas nickase, that recognizes a specific PAM sequence.
  • a nick may be generated by a nickase capable of specifically cleaving one of the two complementary strands of a double-stranded target DNA, or may be generated by a nuclease active Cas protein, e.g., Cas12.
  • a Cas12 protein e.g., Cas12i2 may be capable of generating a nick (or a single stranded break) on each of the two complementary strands of a double-stranded target DNA, wherein the two nicks are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides WSGR Docket No.59761-781.601 apart from each other.
  • the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA.
  • the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA.
  • the nick site is downstream of a PAM sequence recognized by a Cas12i2 nickase or a Cas12i2 nuclease. In some embodiments, the nick site is downstream of the PAM sequence, and the PAM sequence is recognized by a Cas12i protein or domain thereof, e.g., a Cas12i2 protein or a variant thereof as described herein.
  • a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene. In some embodiments, the bound prime editor generates a nick at a nick site of the double-stranded target DNA, e.g., on the PAM strand or the target strand.
  • 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 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 of a PEgRNA is complementary to a sequence in an endogenous 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 prime editor generates a nick at a nick site on the PAM strand of the double stranded target DNA, and the PBS anneals with a free 3’ end formed at the nick site to prime DNA synthesis templated by the editing template.
  • the editing template encodes a single stranded DNA that is identical to the editing target sequence on the PAM strand except for the one or more mismatches at the intended nucleotide edits.
  • the prime editor generates a nick at a nick site on the non-PAM strand of the double stranded target DNA, and the PBS anneals with a free 3’ end formed at the nick site to prime DNA synthesis templated by the editing template.
  • the editing template encodes a single stranded DNA that is identical to the editing target sequence on the non-PAM strand except for the one or more mismatches at the intended nucleotide edits.
  • the newly synthesized single-stranded DNA equilibrates with the editing target sequence on the edited strand of the target gene for pairing with the other, un-edited strand of the target gene.
  • the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1.
  • the FEN is an endogenous FEN, for example, in a cell comprising the target gene.
  • the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided WSGR Docket No.59761-781.601 in trans.
  • the newly synthesized single stranded DNA which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the PAM strand of the target gene.
  • the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene.
  • the newly synthesized single- stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the un-edited strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands.
  • a prime editor of this disclosure is configured to bind a prime editing guide RNA (PEgRNA).
  • a PEgRNA comprises at least one of: a spacer, an extension arm, and a gRNA core.
  • a spacer may comprise a sequence that hybridizes to a first strand of a double stranded target DNA sequence.
  • a spacer may comprise a sequence that is complementary to a first strand of a double stranded target DNA sequence.
  • the spacer may comprise complementary sequence to a search target sequence in the first strand of the double stranded DNA sequence.
  • an extension arm may comprise a sequence that hybridizes to a second strand (i.e., the complementary strand of the first strand) of the double stranded target DNA sequence.
  • an extension arm may comprise a sequence that is complementary to a second strand (i.e., the complementary strand of the first strand) of the double stranded target DNA sequence.
  • a gRNA core may comprise a sequence that interacts with the second polypeptide of the prime editor (i.e., interacts with the DNA binding domain of the prime editor).
  • a PEgRNA may be a single RNA sequence.
  • a spacer, an extension arm, and a gRNA core may be in a single stranded RNA sequence.
  • a spacer, an extension arm, and a gRNA core may be in a single strand of a double stranded RNA sequence.
  • a PEgRNA may comprise a spacer, an extension arm, and a gRNA core in a single RNA sequence.
  • a PEgRNA may comprise a spacer, an extension arm, and a gRNA core in a single RNA sequence in a 5’-3’ orientation.
  • a PEgRNA may comprise a gRNA core, an extension arm, and a spacer in a single RNA sequence in a 3’-5’ orientation.
  • a PEgRNA may comprise a spacer, a gRNA core, and an extension arm are in a single RNA sequence in a 5’-3’ orientation.
  • a PEgRNA may comprise an extension arm, a spacer, and a gRNA core are in a single RNA sequence in 5’-3’ orientation.
  • a PEgRNA may comprise an extension arm, a gRNA core, and a spacer in a single RNA sequence in 5’-3’ orientation.
  • a PEgRNA may comprise a gRNA core, an extension arm, and a spacer in a single RNA sequence in 5’-3’ orientation.
  • a PEgRNA may comprise a gRNA core, a spacer and an extension arm in a single RNA sequence in 5’-3’ orientation.
  • a PEgRNA WSGR Docket No.59761-781.601 may comprise multiple RNA molecules.
  • a spacer, an extension arm, and a gRNA core may be in multiple single stranded RNA sequences.
  • a spacer may be in a single stranded RNA sequence.
  • an extension arm may be in a single stranded RNA sequence.
  • a gRNA core may be in a single stranded RNA sequence.
  • a PEgRNA comprises two or more RNA molecules.
  • a PEgRNA comprises: (i) a first RNA molecule comprising a spacer and a gRNA core, and (ii) a second RNA molecule comprising an extension arm that comprises an editing template and a PBS.
  • a PEgRNA comprises: (i) a first RNA molecule comprising a spacer and a first half of a gRNA core, and (ii) a second RNA molecule comprising a second half of a gRNA core and an extension arm that comprises an editing template and a PBS.
  • a spacer may comprise a sequence that hybridizes to a first strand of a double stranded target DNA sequence.
  • a spacer may comprise a sequence that is complementary to a first strand of a double stranded target DNA sequence.
  • a spacer may hybridize to a first strand of a double stranded target DNA sequence through complementary base pairing of the nucleotides. In some cases, a spacer may hybridize to a search target sequence of a first strand of a double stranded target DNA sequence.
  • a spacer may be 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, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least
  • a spacer is between 10 and 30 nucleotides. In some embodiments, a spacer is between 12 and 24 nucleotides. In some embodiments, a spacer is between 15 and 22 nucleotides. In some embodiments, a spacer is between 16 and 20 nucleotides. In some embodiments, a spacer is between 17 and 22 nucleotides in length. In some embodiments, a spacer is between 18 and 22 nucleotides in length. In some embodiments, a spacer is 20 nucleotides in length. [0112] In some cases, an extension arm of a PEgRNA may comprise a PBS or a DNA synthesis template.
  • an extension arm of a PEgRNA may comprise a PBS and a DNA synthesis template.
  • an extension arm of a PEgRNA comprises, from 5’ to 3’, a DNA synthesis template and a PBS.
  • an extension arm of a PEgRNA may comprise a primer binding site (PBS).
  • a “primer binding site” (also referred to as PBS or primer binding site sequence) is a single-stranded portion of the PEgRNA that comprises a region of complementarity to an endogenous sequence upstream of a nick site in the double-stranded target DNA sequence.
  • the PBS comprises a region of complementarity to the PAM strand (i.e., the non-target 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 a nick site on the PAM strand, e.g., at a nick generated by a Cas12i2 nuclease complexed with the PEgRNA.
  • the PBS comprises region of complementarity to the target strand (i.e. the strand having complementarity to the spacer).
  • the PBS is complementary or substantially complementary to a sequence on the target strand of the double-stranded target DNA that is immediately upstream of a nick site on the target strand, e.g., at a nick generated by a Cas12i2 nuclease complexed with the PEgRNA.
  • 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 a nick site on the target strand and/or the PAM strand of the double stranded target DNA.
  • the PBS is generated at the nick site.
  • the PBS can anneal to a free 3’ end on the target strand of the double stranded target DNA at the nick site. In some embodiments, the PBS can anneal to a free 3’ end on the PAM strand of the double stranded DNA at the nick site. In some embodiments, the at the nick site of a strand of the double-stranded target DNAcan initiate target-primed DNA synthesis.
  • the PBS of a PEgRNA may be 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, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least
  • a PBS is between 5 and 25 nucleotides. In some embodiments, a PBS is between 6 and 20 nucleotides. In some embodiments, a PBS is between 7 and 15 nucleotides. In some embodiments, a PBS is between 8 and 14 nucleotides. In some embodiments, the PBS is about 3-60 nucleotides in length. In some embodiments, the PBS is about 13-60 nucleotides in length. In some embodiments, the PBS is about 13, about 30, or about 60 nucleotides in length. [0115] In some instances, an extension arm of a PEgRNA may comprise a DNA synthesis template (also referred to as an editing template).
  • an “editing template” of a PEgRNA is a single-stranded of the PBS.
  • the editing template encodes one or more nucleotide edits compared to the endogenous sequence of the double-stranded target DNA.
  • the editing template comprises a region of complementarity to an endogenous sequence of the double-stranded target DNA.
  • the editing template comprises a region of complementarity to the PAM strand (i.e., the non-target strand), and WSGR Docket No.59761-781.601 encodes one or more intended nucleotide edits compared to the endogenous sequence of the PAM strand of the double stranded target DNA.
  • the extension arm comprises (i) an editing template having a region of complementarity to and encoding one or more nucleotide edits compared to a sequence immediately downstream of a nick site on the PAM strand of the double- stranded target DNA, and (ii) a PBS having a region of complementarity to a sequence immediately upstream of a nick site on the PAM strand of the double-stranded target DNA.
  • the editing template comprises a region of complementarity to the target strand (i.e., the non-PAM strand), and encodes one or more intended nucleotide edits compared to the endogenous sequence of the target strand of the double stranded target DNA.
  • the extension arm comprises (i) an editing template having a region of complementarity to and encoding one or more nucleotide edits compared to a sequence immediately downstream of a nick site on the target strand of the double-stranded target DNA, and (ii) a PBS having a region of complementarity to a sequence immediately upstream of a nick site on the target strand of the double-stranded target DNA.
  • the one or more nucleotide edits encoded by the DNA synthesis template may comprise any nucleotide changes known in the art.
  • a nucleotide edit comprises one or more insertions, deletions, or substitutions at the intended nucleotide edit positions in the target DNA sequence, or any combinations thereof, as compared to a double stranded target DNA sequence.
  • 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 PAM strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit positions. [0118] - as between the PBS and the editing template, and the relative positions as among elements of a 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 encodes a single stranded DNA, wherein the single stranded DNA has identity or substantial identity to the editing target sequence except for one WSGR Docket No.59761-781.601 or more insertions, deletions, or substitutions at the positions of the one or more intended nucleotide edits.
  • the DNA synthesis template may comprise a portion that is homologous to the double stranded target DNA sequence. In some instances, the DNA synthesis template may be homologous to the double stranded target DNA sequence.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be at least about 85 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 90 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 95 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be at least about 96 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 97 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 98 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be at least about 99 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 99.9 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, or at least about 80 %, absent a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be from about 50 to about 60 %, from about 55 to about 65 %, from about 60 to about 70 %, from about 65 to about 75 %, from about 70 to about 80 %, from about 75 to about 85 %, from about 80 to about 90 %, from about 85 to about 95 %, or from about 90 to about 100 %, absent a nucleotide edit in the DNA synthesis template.
  • the DNA synthesis template may be homologous to the double stranded target DNA sequence.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be at least about 85 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 90 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 95 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded WSGR Docket No.59761-781.601 target DNA sequence may be at least about 96 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 97 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 98 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be at least about 99 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 99.9 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence in some cases, may be at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, or at least about 80 %, with a nucleotide edit in the DNA synthesis template.
  • the homology between the DNA synthesis template and the double stranded target DNA sequence may be from about 50 to about 60 %, from about 55 to about 65 %, from about 60 to about 70 %, from about 65 to about 75 %, from about 70 to about 80 %, from about 75 to about 85 %, from about 80 to about 90 %, from about 85 to about 95 %, or from about 90 to about 100 %, with a nucleotide edit in the DNA synthesis template.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 85% sequence identity to a strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 90 % sequence identity to a strand of a target DNA sequence. In some cases, the DNA synthesis template may comprise a nucleotide sequence comprising at least about 95 % sequence identity to a strand of a target DNA sequence. In some cases, the DNA synthesis template may comprise a nucleotide sequence comprising at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99% sequence identity to a strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 86 %, at least about 87 %, at least about 88 %, or at least about 89 % sequence identity to a strand of a target DNA sequence. In some cases, the DNA synthesis template may comprise a nucleotide sequence comprising at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, or at least about 80 % sequence identity to a strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 85% sequence identity to a first strand of a target DNA sequence. In some cases, the DNA synthesis template may comprise a nucleotide sequence comprising at least about 90 % sequence identity to a first strand of a target DNA sequence. In some cases, the DNA synthesis template may comprise a nucleotide sequence comprising at least about 95 % sequence identity to a first strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 96 %, at least about 97 %, at least about 98 %, or at WSGR Docket No.59761-781.601 least about 99% sequence identity to a first strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 86 %, at least about 87 %, at least about 88 %, or at least about 89 % sequence identity to a first strand of a target DNA sequence.
  • the DNA synthesis template may comprise a nucleotide sequence comprising at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, or at least about 80 % sequence identity to a first strand of a target DNA sequence.
  • Dual Prime Editing involves 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”).
  • 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 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. In some embodiments, each of the two PEgRNAs anneals with a separate search target sequence through its spacer.
  • references to a “PAM strand”, a “non-PAM strand”, a “target strand’, a “non- target strand”, are relative in the context of a specific PEgRNA, e.g., one of the two PEgRNAs in dual prime editing.
  • 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.
  • the two PEgRNAs each can direct a prime editor to initiate the prime editing process on the two complementary strands of the target DNA.
  • a first PEgRNA comprises a first spacer complementary to a first search target sequence on a first strand of a double-stranded target DNA.
  • 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, which is complementary to the first target strand.
  • 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.
  • dual prime editing of a double stranded target DNA involves a prime editing system that comprises a first PEgRNA and a second PEgRNA.
  • Each of the two PEgRNAs in WSGR Docket No.59761-781.601 dual prime editing may comprise an extension arm that comprises a primer binding site and an editing template.
  • the extension arms can have complementarity to either strand of the double stranded target DNA.
  • the first PEgRNA comprises a spacer that comprises a region of complementarity to a first search target sequence on a first strand of the double-stranded target DNA, a gRNA core capable of complexing with a DNA binding domain of a first prime editor, and a first extension arm that comprises a first editing template and a first PBS, wherein the first PBS comprises a region of complementarity to a sequence upstream of a first nick site in a second strand of the double-stranded target DNA that is complementary to the first strand.
  • the second PEgRNA comprises a spacer that comprises a region of complementarity to a second search target sequence in the second strand of the double-stranded target DNA, a gRNA core capable of binding to a DNA binding domain of a second prime editor, and a second extension arm that comprises a second editing template and a second PBS, wherein the second PBS comprises a region of complementarity to a sequence upstream of a second nick site in the first strand of the double- stranded target DNA.
  • the first PEgRNA complexes with the first prime editor and generates a first nick at the first nick site in the second strand of the double-stranded target DNA, the first PBS anneals with a free 3’ end formed at the first nick site, and the first prime editor initiates DNA synthesis from the first nick site to generate a first single stranded DNA encoded by the first editing template.
  • the second PEgRNA complexes with the second prime editor and generates a second nick at the second nick site in the first strand of the double-stranded target DNA, the second PBS anneals with a free 3’ end formed at the second nick site, and the second prime editor initiates DNA synthesis from the second nick site to generate a second single stranded DNA encoded by the first editing template.
  • An exemplary dual prime editing schematic is provided in FIG.4A.
  • the first PEgRNA comprises a spacer that comprises a region of complementarity to a first search target sequence on a first strand of the double-stranded target DNA, a gRNA core capable of binding to a DNA binding domain of a first prime editor, and a first extension arm that comprises a first editing template and a first PBS, wherein the first PBS comprises a region of complementarity to a sequence upstream of a first nick site in the first strand of the double-stranded target DNA.
  • the second PEgRNA comprises a spacer that comprises a region of complementarity to a second search target sequence on the second strand of the double-stranded target DNA that is complementary to the first strand, a gRNA core capable of binding to a DNA binding domain of a second prime editor, and a second extension arm that comprises a second editing template and a second PBS, wherein the second PBS comprises a region of complementarity to a sequence upstream of a second nick site in the second strand of the double- stranded target DNA.
  • the first PEgRNA complexes with the first prime editor and generates a first nick at the first nick site in the first strand of the double-stranded target DNA, the WSGR Docket No.59761-781.601 first PBS anneals with a free 3’ end formed at the first nick site, and the first prime editor initiates DNA synthesis from the first nick site to generate a first single stranded DNA encoded by the first editing template.
  • the second PEgRNA complexes with the second prime editor and generates a second nick at the second nick site in the second strand of the double-stranded target DNA, the second PBS anneals with a free 3’ end formed at the second nick site, and the second prime editor initiates DNA synthesis from the second nick site to generate a second single stranded DNA encoded by the first editing template.
  • An exemplary dual prime editing schematic is provided in FIG.4B.
  • the first nick site is downstream of the first search target sequence.
  • the second nick site is downstream of the second search target sequence.
  • the endogenous sequence of the double stranded DNA between the first nick and the second nick (referred to as the inter-nick duplex, IND) is replaced by the first and the second single stranded DNA encoded by the first and second editing template, and the one or more nucleotide edits encoded by the editing templates are thereby incorporated into the target double stranded DNA.
  • the nucleotide edits encoded by the first and/or the second editing template can be one or more nucleotide substitutions, one or more insertions, one or more deletions, or any combination thereof.
  • the first editing template comprises a region of complementarity to the second editing template.
  • the first editing template comprises a region of complementarity at its 5' end (corresponding to the 3’ end of the first newly synthesized singles stranded DNA) to the second editing template, e.g., a region of complementarity of 5, 10, 15, 20, 25, 30 or more nucleotides.
  • the second editing template comprises a region of complementarity at its 5' end (corresponding to the 3’ end of the second newly synthesized singles stranded DNA) to the first editing template, e.g., a region of complementarity of 5, 10, 15, 20, 25, 30 or more nucleotides.
  • the first editing template comprises a region of identity to a sequence of the IND.
  • the second editing template comprises a region of identity to a sequence of the IND.
  • the first template comprises a region of identity to an endogenous sequence of the double stranded target DNA upstream or downstream of the IND.
  • the second template comprises a region of identity to an endogenous sequence of the double stranded target DNA upstream or downstream of the IND. Exemplary schematics of single stranded DNAs encoded by the first and second editing templates as well as complementarity/homology are provided in FIG.s 4C-4H.
  • the first prime editor and the second prime editor each comprise a programmable DNA binding domain capable of generating nicks or double stranded breaks in the double stranded target DNA.
  • the first prime editor and the second prime editor comprises the same programmable DNA binding domain.
  • the programmable DNA binding domain(s) of the prime editor in dual prime editing may be nickases or active nucleases.
  • the first and the second prime editor each comprises a Cas12 nickase that specifically nicks the PAM strand (and not the target strand).
  • Such prime editors may be used for dual WSGR Docket No.59761-781.601 prime editing with a pair of PEgRNAs that comprise a first PEgRNA and a second PEgRNA, wherein the first PEgRNA comprises a first spacer having a region of complementarity to a first strand of the double-stranded target DNA and a first extension arm comprising a first PBS and a first editing template, wherein the first PBS comprises a region of complementarity to the sequence in the second strand of the double-stranded target DNA upstream of the first nick generated by the Cas12 nickase, and wherein the second PEgRNA comprises a second spacer having a region of complementarity to the second strand of the double-stranded target DNA and a second extension arm comprising a second editing template and a second PBS, wherein the second PBS comprises a region of complementarity to the sequence in the first strand of the double-stranded target DNA upstream of the second nick generated by the Ca
  • the Cas12 nickase is a Cas12i2 nickase.
  • the first and the second prime editor each comprises a Cas12 nickase that specifically nicks the target strand (and not the PAM strand).
  • Such prime editors may be used for dual prime editing with a pair of PEgRNAs that comprise a first PEgRNA and a second PEgRNA, wherein the first PEgRNA comprises a first spacer having a region of complementarity to a first strand of the double-stranded target DNA and a first extension arm comprising a first editing template and a first PBS, wherein the first PBS comprises a region of complementarity to the sequence in the first strand of the double-stranded target DNA upstream of the first nick generated by the Cas12 nickase, and wherein the second PEgRNA comprises a second spacer having a region of complementarity to the second strand of the double-stranded target DNA and a second extension arm comprising a second editing template and a second PBS, wherein the second PBS comprises a region of complementarity to the sequence in the second strand of the double-stranded target DNA upstream of the second nick generated by the Cas12 nickase.
  • the Cas12 nickase is a Cas12i2 nickase.
  • the first and/or prime editor comprises a fully active nuclease, e.g., a Cas nuclease, that is capable of cleaving both strands of the double-stranded target DNA.
  • the prime editor(s) may be used for dual prime editing with a pair of PEgRNAs that comprises a first PEgRNA and a second PEgRNA, wherein (i) the first PEgRNA comprises a first spacer having a region of complementarity to a first strand of the double-stranded target DNA and a first extension arm comprising a first editing template and a first PBS, wherein the first PBS comprises a region of complementarity to the sequence in the second strand of the double-stranded target DNA upstream of the first nick generated by the Cas nuclease, and wherein the second PEgRNA comprises a second spacer having a region of complementarity to the second strand of the double-stranded target DNA and a second a second extension arm comprising a second editing template and a second PBS, wherein the second PBS comprises a region of complementarity to the sequence in the first strand of the double-stranded target DNA upstream of the second nick generated by the Cas nuclease,
  • the Cas nuclease is a Cas12i nuclease, for example, a Cas12i1, Cas12i2, Cas12i3, or Cas12i4 nuclease.
  • the Cas nuclease is a Cas12i2 nuclease or a variant provided herein or known in the art.
  • the Cas12i2 nuclease comprises an amino acid sequence of at least about 85%, 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1017.
  • dual prime editing involves a pair of PEgRNAs each complexed with a prime editor that comprises a fully active nuclease capable of cleaving both strands of the double- stranded target DNA.
  • the pair of PEgRNAs and the prime editor may be designed such that the prime editor cleaves the double-stranded target DNA at the same positions when complexed with the first PEgRNA and when complexed with the second PEgRNA.
  • the prime editor comprises a Cas nuclease, e.g., a Cas12 nuclease, which is capable of cleaving the first strand and the second strand of the double-stranded target DNA at positions 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from each other (referred to as staggered cuts).
  • a Cas nuclease e.g., a Cas12 nuclease, which is capable of cleaving the first strand and the second strand of the double-stranded target DNA at positions 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from each other (referred to as staggered cuts).
  • a first PEgRNA comprising a first spacer having a region of complementarity to a first strand of the double-stranded target DNA may complex with the prime editor and direct the Cas12 nuclease to cleave the first strand at a first nick site and cleave the second strand complementary to the first strand at a second nick site 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from the first nick site.
  • a second PEgRNA comprising a second spacer may be designed to have a region of complementarity to the second strand of the double-stranded target DNA, which may complex with the prime editor and direct the Cas12 nuclease to cleave the second strand at the second nick site and the first strand at the first nick site, which are the same as the nick site generated by the prime editor when complexed with the first PEgRNA.
  • a dual prime editing system while involving an active nuclease, may only generate one double stranded break (or two nicks) on the target DNA and may thus be advantageous.
  • the Cas12 nuclease is a Cas12i nuclease, for example, a Cas12i1, Cas12i2, Cas12i3, or Cas12i4 nuclease.
  • the Cas nuclease is a Cas12i2 nuclease or a variant provided herein or known in the art.
  • the Cas12i2 nuclease comprises an amino acid sequence of at least about 85%, 86%, 87%, 88%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1017.
  • the first PEgRNA and the second PEgRNA in a dual prime editing system may each be in a single RNA molecule, or may be provided in two or more RNA molecules.
  • dual prime editing involves a prime editing system comprising (i) a first spacer and a first gRNA core, (ii) a first extension arm comprising, from 5’ to 3’, a first editing template and a first PBS, (iii) a second spacer and a second gRNA core, and (iv) a second extension arm comprising, from 5’ to 3’, a second editing template and a second PBS.
  • prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the first spacer, the first gRNA core, and the first extension arm. In some embodiments, prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the first gRNA core, the first spacer, and the first extension arm. In some embodiments, the prime editing system comprises (i) a RNA molecule comprising, from 5’ the 3’, the first spacer and the first gRNA core, and (ii) a separate RNA molecule comprising the first extension arm.
  • the prime editing system comprises (i) a RNA molecule comprising, from 5’ the 3’, the first gRNA core and the first spacer, and (ii) a separate RNA molecule comprising the first extension arm.
  • the prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the first spacer, the first gRNA core, and the second extension arm.
  • prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the first gRNA core, the first spacer, and the second extension arm.
  • prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the second spacer, the second gRNA core, and the second extension arm. In some embodiments, prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the second gRNA core, the second spacer, and the second extension arm. In some embodiments, the prime editing system comprises (i) a RNA molecule comprising, from 5’ the 3’, the second spacer and the second gRNA core, and (ii) a separate RNA molecule comprising the second extension arm.
  • the prime editing system comprises (i) a RNA molecule comprising, from 5’ the 3’, the second gRNA core and the second spacer, and (ii) a separate RNA molecule comprising the second extension arm.
  • the prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the second spacer, the second gRNA core, and the first extension arm.
  • prime editing system comprises a RNA molecule comprising, from 5’ to 3’, the second gRNA core, the second spacer, and the first extension arm.
  • Prime Editor As used herein, the term “prime editor”, “PE” or “prime editor protein” refers to the polypeptide or polypeptide components involved in prime editing.
  • Prime editors described herein may comprise multiple polypeptides or protein domains.
  • a prime editor includes a polypeptide domain having DNA binding activity (e.g., a DNA binding domain).
  • a prime editor comprises a polypeptide that comprises a DNA binding domain.
  • a prime editor includes a polypeptide domain having DNA polymerase activity (e.g., a DNA polymerase domain).
  • a prime editor comprises a polypeptide that WSGR Docket No.59761-781.601 comprises a DNA polymerase domain.
  • a prime editor comprises a polypeptide domain having DNA binding activity (e.g., a DNA binding domain), and a polypeptide domain having DNA polymerase activity (e.g., a DNA polymerase domain).
  • a prime editor comprises a polypeptide that comprises a DNA binding domain and a polypeptide that comprises a DNA polymerase domain.
  • the prime editor further comprises a polypeptide domain having a nuclease activity.
  • the polypeptide domain having the nuclease activity comprises a nickase, or a fully active nuclease.
  • the DNA binding domain comprises a nuclease domain or nuclease activity.
  • the nuclease domain is a nickase, or a fully active nuclease.
  • the prime editor comprises a polypeptide domain that is an inactive nuclease.
  • the DNA binding domain comprises a nuclease domain that is an inactive nuclease.
  • the polypeptide domain having DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas12i nickase or a Cas12i nuclease.
  • the DNA binding domain is a nucleic acid guided DNA binding domain for example, a CRISPR-Cas protein, for example, a Cas12i2 nickase or a Cas12i2 nuclease.
  • the DNA binding domain is a Cas12i2 protein domain.
  • the Cas12i2 protein domain comprises a nickase or comprises a nickase activity.
  • the Cas12i2 protein or Cas12i2 protein domain comprises at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1017.
  • the Cas12i2 protein or Cas12i2 protein domain comprises at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1017.
  • the amino acid sequence of the Cas12i2 protein is selected from any one of SEQ ID NOs: 1018, 1019, 1020, 1021 or 1022 or a protein domain derived from any one of such Cas12i2 proteins.
  • the amino acid sequence of the Cas12i2 protein is selected from either of SEQ ID NOs: 1019 or 1022.
  • the Cas12i protein or Cas12i protein domain is Cas12i1, Cas12i3 or Cas12i4 or a protein domain derived from any one of such Cas12i proteins.
  • the amino acid sequence of the Cas12i protein or Cas12i protein domain comprises at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 1023, 1024, 1025 or 1026.
  • the amino acid sequence of the Cas12i protein or Cas12i protein domain comprises at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 1023, 1024, 1025 or 1026. In some embodiments, the amino acid sequence of the Cas12i protein or Cas12i protein domain comprises at least 95%, 96%, 97%, 98%, WSGR Docket No.59761-781.601 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 1023, 1024, 1025 or 1026. In some embodiments, the amino acid sequence of the Cas12i protein is selected from any one of SEQ ID NOs: 1023, 1024, 1025 or 1026.
  • 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 binding domain comprises a template-dependent DNA polymerase for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA polymerase domain comprises an RNA-dependent DNA polymerase that is a reverse transcriptase domain (RT domain) or a reverse transcriptase (RT).
  • RT domain reverse transcriptase domain
  • RT reverse transcriptase
  • RT reverse transcriptase
  • RT reverse transcriptase
  • a prime editor comprises reverse transcriptase (RT) activity.
  • the first polypeptide of the prime editor may have activity for target primed reverse transcription.
  • the polypeptide domain having DNA polymerase activity comprises a reverse transcriptase activity (e.g., activity for target primed reverse transcription).
  • the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having a 5’ endonuclease activity, e.g., a 5' endogenous DNA flap endonuclease (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.
  • the recombinase may be a serine recombinase (e.g., a resolvase or an invertase). In some embodiments, the recombinase may be a tyrosine recombinase (e.g., an integrase).
  • a prime editor system may insert one or more SSR recognition sequences into the double stranded DNA target. [0142] A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, 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 and a reverse transcriptase polypeptide that are derived from WSGR Docket No.59761-781.601 different species.
  • a prime editor may comprise a Cas12i2 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.
  • M-MLV Moloney murine leukemia virus
  • a prime editor comprises one or more polypeptide domains (e.g., a DNA binding domain and a DNA polymerase domain) provided in trans as separate proteins.
  • a prime editor comprises one or more polypeptide domains provided 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 can comprise a DNA binding domain and a DNA polymerase domain (e.g., a reverse transcriptase domain) as separate proteins associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which can, in some embodiments, be linked to a PEgRNA.
  • a prime editor can comprise a DNA binding domain and a DNA polymerase domain (e.g., a reverse transcriptase domain) as separate proteins associated with each other by peptide recruitment sequences on one or both proteins (e.g., leucine zipper sequences on both proteins).
  • a prime editor can comprise a DNA binding domain, a DNA polymerase domain and a recombinase as separate proteins.
  • polypeptide domains of a prime editor e.g., a DNA binding domain and a DNA polymerase domain
  • a prime editor can comprise a DNA binding domain and a DNA polymerase domain (e.g., a reverse transcriptase domain) fused or linked with each other by a peptide linker (e.g., linkers disclosed set forth in SEQ ID NOs: 272-318 or 1014-1016).
  • a prime editor can comprise a DNA binding domain, a DNA polymerase domain and a recombinase as a fusion protein.
  • Prime editor polypeptide components can be encoded by one or more polynucleotides in whole or in part.
  • the present disclosure contemplates polynucleotides encoding the prime editor components, for example, a polynucleotide encoding a DNA binding domain, and a polynucleotide encoding a DNA polymerase domain.
  • the present disclosure also contemplates a single polynucleotide comprising a polynucleotide encoding a DNA binding domain, and a polynucleotide encoding a DNA polymerase domain.
  • the polynucleotide encoding a DNA binding domain, and a polynucleotide encoding a DNA polymerase domain are linked by a linker polynucleotide to result in a fusion protein comprising the DNA polymerase domain and DNA binding domain linked by a linker.
  • the polynucleotide encodes a fusion protein comprising additional polypeptide domains, e.g., a recombinase.
  • 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.
  • a prime editor fusion protein can 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 a vector, e.g., an AAV vector.
  • components of a prime editor disclosed herein may be brought together post-translationally via a split-intein.
  • a prime editor comprises a DNA polymerase domain and a DNA binding domain wherein the amino acid sequences of the DNA polymerase domain and the DNA binding domain comprise a N-terminal methionine.
  • a prime editor comprises a DNA polymerase domain and a DNA binding domain wherein the amino acid sequences of the DNA polymerase domain and the DNA binding domain do not comprise a N-terminal methionine. In some embodiments, a prime editor comprises a DNA polymerase domain and a DNA binding domain wherein the amino acid sequence of the DNA polymerase domain comprises a N-terminal methionine and the amino acid sequence of the DNA binding domain does not comprise a N-terminal methionine.
  • a prime editor comprises a DNA polymerase domain and a DNA binding domain wherein the amino acid sequence of the DNA polymerase domain does not comprise a N- terminal methionine and the amino acid sequence of the DNA binding domain comprises a N-terminal methionine.
  • a prime editor component thereof e.g., a polypeptide comprising a DNA binding domain and/or a polypeptide comprising a DNA polymerase domain
  • 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 can comprise a Cas12i2 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.
  • M-MLV Moloney murine leukemia virus
  • An RT or an RT domain may be rationally engineered, in some embodiments. Such an engineered RT or RT domain may comprise sequences or amino acid changes different from a naturally occurring RT or RT domain.
  • a prime editor comprises a polypeptide domain (e.g., a DNA polymerase domain) comprising a DNA polymerase activity.
  • the prime editor comprises a polypeptide that comprises a DNA polymerase domain.
  • a prime editor comprises a polynucleotide that encodes a polymerase domain, e.g., a DNA polymerase domain.
  • the DNA polymerase domain can be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, a wild type DNA polymerase, a full-length DNA polymerase, or WSGR Docket No.59761-781.601 can be a functional mutant, a functional variant, or a functional fragment thereof.
  • the DNA polymerase domain is a template dependent DNA polymerase domain.
  • the DNA polymerase can 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.
  • the DNA polymerase domain is 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 can be a chimeric or hybrid PEgRNA, and comprising an extension arm comprising a DNA strand.
  • the chimeric or hybrid PEgRNA can 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 prime editors provided herein comprises a DNA polymerase domain comprising an amino acid sequence that does not a have a N-terminus methionine.
  • the prime editors provided herein comprises a DNA polymerase domain comprising an amino acid sequence comprising a N-terminus methionine.
  • the amino acid sequence of a DNA polymerase domain may be N-terminally modified by one or more processing enzymes, e.g., by Methionine aminopeptidases (MAP).
  • MAP Methionine aminopeptidases
  • the DNA polymerase domain can be a wild type DNA polymerase, for example, from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes.
  • the DNA polymerases can be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like.
  • the DNA 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 a 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. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is a E.coli Pol IV DNA polymerase. [0151] In some embodiments, the DNA polymerase comprises a eukaryotic DNA polymerase. In some embodiments, the DNA polymerase is a Pol-beta DNA polymerase, a Pol-lambda DNA WSGR Docket No.59761-781.601 polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase.
  • the DNA polymerase is a Pol-alpha DNA polymerase. In some embodiments, the DNA polymerase is a POLA1 DNA polymerase. In some embodiments, 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.
  • the DNA polymerase is a POLD3 DNA polymerase. In some embodiments, the DNA polymerase is a POLD4 DNA polymerase. In some embodiments, 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.
  • POLH Pol-eta
  • POLI Pol-iota
  • the DNA polymerase is a Pol-kappa (POLK) DNA polymerase. In some embodiments, 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.
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • 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.
  • poly 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 WSGR Docket No.59761-781.601 polymerase. In some embodiments, the DNA Polymerase is a Pol III family DNA polymerase. In 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. In some embodiments, the Pol I DNA activity.
  • 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).
  • Reverse Transcriptases [0156]
  • a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT).
  • the DNA polymerase domain is an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT).
  • the DNA polymerase domain is a reverse transcription (RT) domain, for example, a reverse transcriptase (RT).
  • RT reverse transcription
  • An RT or an RT domain can 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, a full length RT, a functional mutant, a functional variant, or a functional fragment thereof; 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. In some embodiments, the engineered RT may have improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity. In some embodiments, a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT. In some embodiments, a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a PE2 RT reference. [0157] In some embodiments, a prime editor comprises a virus RT, for example, a retrovirus RT.
  • the RT is a virus RT, for example, a retrovirus RT.
  • virus RT include Moloney murine leukemia virus (M-MLV) RT; 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 chain A, HIV RT chain B, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) 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
  • M-MLV Mol
  • the RT is selected from the RT of WSGR Docket No.59761-781.601 Feline Leukemia Virus RT (Accession No. NP955579.1), HIV-1 chain A RT (Martinelli et al. Virology 1990, 174:135-144), HIV-1 chain B RT (Stammers et al. J. Mol. Biol.1994, 242: 586-588), RSV RT (Accession No. ACL14945), Cauliflower Mosaic Virus RT (Farzadfar et al. Virus Genes 2013, 47:347-356), Klebsiella pneumonia (Accession No. RFF81513.1), E. coli RT (Accession No.
  • the prime editor comprises a wild type M-MLV RT.
  • the RT domain or RT is a wild type M-MLV RT.
  • An exemplary sequence of a wild type M-MLV RT is provided in SEQ ID NO: 857.
  • the prime editor comprises a reference M-MLV RT. The sequence of a reference M-MLV RT is disclosed in SEQ ID NO: 855.
  • the prime editor comprises an M-MLV RT that comprises one or more amino acid substitutions.
  • the prime editor comprises a truncated M-MLV RT.
  • the prime editor comprises a M-MLV RT as set forth in SEQ ID NO: 857. In some embodiments, the prime editor comprises a M-MLV RT as set forth in SEQ ID NO: 856. In some embodiments, the prime editor comprises a M-MLV RT as set forth in SEQ ID NO: 855. In some embodiments, the prime editor comprises a M-MLV RT as set forth in SEQ ID NO:884.
  • the prime editor comprises a M-MLV RT that comprises one or more of amino acid substitutions P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to a reference M-MMLV RT as set forth in SEQ ID NO:855, where X is any amino acid other than the reference amino acid.
  • the prime editor comprises a M-MLV RT that comprises one or more of amino acid substitutions P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N as compared to the reference M-MLV RT as set forth in SEQ ID NO: 855.
  • prime editor comprises one or more amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 855.
  • the prime editor comprises a M-MLV RT that comprises amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 855.
  • the RT is an M-MLV RT that comprises one or more of amino acid substitutions P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to the reference M-MLV RT as set forth in SEQ ID NO: 857, where X is any amino acid other than the wild type amino acid.
  • the RT is an M-MMLV RT that comprises one or more of amino acid substitutions P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, P448A, D449G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N as compared to the reference M-MMLV RT as set forth in SEQ ID NO: 857.
  • the RT is a M-MLV RT that WSGR Docket No.59761-781.601 comprises one or more amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 857.
  • the RT is an M-MLV RT that comprises amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 857.
  • the RT is a truncated M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 857.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 1-95, 129-136, 198- 271, 319-493, 533-846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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% identical to any one of the sequences listed in any of the Tables 1, 2, 3, 7, 10 or 15.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any one of the amino acid sequences listed in any of the Tables 1, 2, 3, 7, 10, or 15.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences listed in in any of the Tables 1, 2, 3, 7, 10, or 15.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 1-95, 129-136, 198- 271, 319-493, 533-846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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 WSGR Docket No.59761-781.601 least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of the sequences set forth in SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 204, 230, 232-244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 489, 990-1006, 209, 210, 231, 229, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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% identical to any one of the sequences listed in any of the Tables 1, 2, 3, 7, 10, or 15.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 204, 230, 232-244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 489, 990-1006, 209, 210, 231, 229.1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any one of the amino acid sequences listed in any of the Tables 1, 2, 3, 7 or 10.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533- 846, 855-857, 884, 990-1006, or 1059-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 204, 230, 232-244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 489, 990-1006, 209, 210, 231, 229, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences listed in in any of the Tables 1, 2, 3, 7, 10, or 15.
  • a prime editor may comprise a RT domain having an amino acid sequence with at least 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% identical to the sequences set forth in any one of SEQ ID NOs: 16, 18, 261 or 270.
  • a prime editor may comprise a RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to the amino acid sequences set forth in SEQ ID NO: 16, 18, 261 or 270.
  • a prime editor may comprise a RT domain having an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 16, 18, 261 or 270. [0164] In some embodiments, a RT domain may comprise an ancestral RT sequence.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT sequence with at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 81-95.
  • a RT domain may comprise an ancestral RT sequence.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT sequence with at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 81, 82, 84, 91, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT sequence with at least 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% identical to any one of the sequences listed in Table 3 or Table 15.
  • an ancestral RT sequence with at least 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% identical to any one of the sequences listed in Table 3 or Table 15.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 81-95 or 1059-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 81, 82, 84, 91, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain, having an amino acid sequence, e.g., an ancestral RT 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any one of the amino acid sequences listed in Table 3 or Table WSGR Docket No.59761-781.601 15.
  • a prime editor may comprise a RT domain, having an amino acid sequence, e.g., an ancestral RT sequence identical to any one of the sequences set forth in SEQ ID NOs: 81-95 or 1059-1109.
  • a prime editor may comprise a RT domain, having an amino acid sequence, e.g., an ancestral RT sequence identical to any one of the sequences set forth in SEQ ID NOs: 81, 82, 84, 91, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., an ancestral RT sequence identical to any one of the sequences listed in Table 3 or Table 15.
  • a prime editor may comprise a RT domain that is a Cas-RT.
  • the RT domains works with Cas1, Cas6, or Cas3 in RNA spacer acquisition.
  • a prime editor may comprise a RT domain, e.g., Cas-RT domain.
  • the Cas1 domain of Cas1-RT-Cas1 may be replaced with a Cas12i2 domain and optionally a linker sequence.
  • a prime editor may comprise a RT domain, e.g., a Cas-RT domain having an amino acid sequence, with at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 129-136, 345, 368, 396, or 533-846.
  • a prime editor may comprise a RT domain e.g., a Cas-RT domain having an amino acid sequence with at least 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% identical to any one of the sequences listed in Tables 1, 2, 7, or 10.
  • a RT domain e.g., a Cas-RT domain having an amino acid sequence with at least 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% identical to any one of the sequences listed in Tables 1, 2, 7, or 10.
  • a prime editor may comprise a RT domain, e.g., a Cas-RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 129-136, 345, 368, 396, 533-846, 855-857 or 884.
  • a prime editor may comprise a RT domain, e.g., a Cas-RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences, e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid substitutions compared to any one of the amino acid sequences listed in any of the Tables 1, 2, 7, and/or 10.
  • a RT domain e.g., a Cas-RT domain having 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences, e.g., mutations e.g., amino acid deletions, amino acid insertions, and/or amino acid
  • a prime editor may comprise a RT domain, e.g., a Cas-RT domain, having an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 129-136, 345, 368, 396, 533-846, 855-857 or 884.
  • a prime editor may comprise a RT domain having an amino acid sequence, e.g., ancestral RT sequence identical to any one of the sequences listed in any of the Tables 1, 2, 7, and/or 10.
  • a DNA polymerase domain e.g., a reverse transcriptase domain may comprise one or more mutations.
  • Mutant reverse transcriptases can, for example, be obtained by WSGR Docket No.59761-781.601 mutating the gene or genes encoding the reverse transcriptase of interest by site-directed or random mutagenesis.
  • the mutation may include a deletion mutation, a point mutation, a substitutional mutation and/or an insertional mutation.
  • the mutation increases the efficiency of the DNA polymerase domain, e.g., a reverse transcriptase domain, e.g., by increasing editing efficiency, e.g., by increasing reverse transcriptase activity, e.g., by increasing stability (e.g., thermostability).
  • the mutated DNA polymerase domain e.g., the mutated RT domain may show 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 efficiency compared to an unmutated DNA polymerase domain, e.g., RT domain.
  • the mutated DNA polymerase domain e.g., the mutated RT domain may show 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% increased activity compared to an unmutated DNA polymerase domain, e.g., RT domain.
  • the prime editor comprises a DNA polymerase domain, e.g., a reverse transcriptase domain that is modified, e.g., by insertion, deletion, or substitution.
  • the modified DNA polymerase domain e.g., a reverse transcriptase domain includes one or more amino acid mutations that are located outside the catalytic domains of the polymerase, e.g., reverse transcriptase.
  • the modified polymerase e.g., reverse transcriptase
  • comprises amino acid mutations e.g., amino acid substitutions, deletions, insertions, or chemical modifications located at any position other than the invariant residues, e.g., conserved catalytic residues.
  • the conserved catalytic residue is an aspartate amino acid, e.g., catalytic aspartate amino acid.
  • the catalytic aspartate amino acid is involved in incorporation of the correct nucleotide.
  • mutating an invariant residue results in 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% loss of DNA polymerase, e.g., reverse transcriptase function. In some embodiments, mutating an invariant residue results in 100% loss of DNA polymerase, e.g., reverse transcriptase function.
  • the amino acid sequence of a DNA polymerase e.g., reverse transcriptase may be aligned with the amino acid sequence of the reference M-MLV RT (SEQ ID NO: 855) to identify a conserved catalytic residue present in the DNA polymerase, e.g., reverse transcriptase (Table 23).
  • the amino acid sequence of a reverse transcriptase e.g., a reference M- MLV RT, e.g., SEQ ID NO: 855, may comprise one or more of D150, D224, and/or D225 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase may comprise one or more of conserved catalytic residues, e.g., conserved aspartate catalytic residues at positions relative to amino acid residues D150, D225, and/or D225 in a corresponding reference M- WSGR Docket No.59761-781.601 MLV RT (SEQ ID NO: 855).
  • the amino acid sequence of a reverse transcriptase e.g., a retron_b7, e.g., SEQ ID NO: 18 may comprise one or more of D113, D191, and/or D192 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a Retron_C10, e.g., SEQ ID NO: 16 may comprise one or more of D72, D159, and/or D160 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase, e.g., a spuma_C4, e.g., SEQ ID NO: 261 may comprise one or more of D152, D214, and/or D215 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a spuma_E3, e.g., SEQ ID NO: 270 may comprise one or more of D152, D156, D214, and/or D215 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a SALVI, e.g., as exemplified in SEQ ID NO: 1004
  • the amino acid sequence of a reverse transcriptase may comprise one or more of D71, D158, and/or D159 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a N112.SENR, e.g., SEQ ID NO: 1096 may comprise one or more of D67, D152, and/or D153conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a N121.SENR, e.g., SEQ ID NO: 1097 may comprise one or more of D68, D155, and/or D156 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a N163.SENR, e.g., SEQ ID NO: 1105 may comprise one or more of D72, D159, and/or D160 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase e.g., a N79.MMLV, e.g., SEQ ID NO: 1068 may comprise one or more of D149, D223, and/or D224 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase, e.g., a N80.MMLV, e.g., SEQ ID NO: 1063 may comprise one or more of D148, D222, and/or D223 conserved catalytic residues.
  • the amino acid sequence of a reverse transcriptase may comprise one or more of D149, D223, and/or D224 conserved catalytic residues.
  • Table 23 shows exemplary conserved catalytic amino acid residues for some reverse transcriptase domains.
  • a prime editor comprises a reverse transcriptase variant derived from a reverse transcriptase shown in Table 23 and comprise one or more amino acid substitutions compared to the reverse transcriptase in Table 23, wherein the one or more amino acid substitutions does not include a substitution at a conserved catalytic residue shown in Table 23.
  • the RT variant may be a functional fragment of a reference RT (e.g., a RT set forth in SEQ ID NO: 855, or an RT domain, for example, provided in Tables 1, 2, 3, 7, and 10) 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 amino acid changes (e.g., amino acid substitution and/or amino acid deletion) compared to a reference RT, (e.g., a RT set forth in SEQ ID NO: 855, or a RT set forth in SEQ ID NO: 856, or a RT domain, for example, provided in Tables 1, 2, 3, 7, and 10).
  • a reference RT e.g., a RT set forth in SEQ ID NO: 855, or an RT domain, for example
  • the RT variant comprises a fragment of a reference RT, e.g., a RT set forth in SEQ ID NO: 855, a RT set forth in 856, or an RT domain, for example, provided in Tables 1, 2, 3, 7, and 10, such that the fragment is at least about 50% identical, 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 corresponding fragment of the reference RT e.g., a RT set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or an RT domain, for example, provided in Table 1, 2, 3, 4, 7, and 10.
  • a reference RT e.g., a RT set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or an RT domain, for example, provided in Table 1, 2, 3,
  • the fragment is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical, 96%, 97%, 98%, 99%, or 99.5% of the amino acid length of a reference sequence, e.g., M-MLV RT set forth in set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or an RT provided in Tables 1, 2, 3, 4, 7, and 10.
  • a reference sequence e.g., M-MLV RT set forth in set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or an RT provided in Tables 1, 2, 3, 4, 7, and 10.
  • WSGR Docket No.59761-781.601 In some embodiments, the RT functional fragment is at least 100 amino acids in length.
  • the RT functional fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or up to 700 amino acids in length.
  • a RT variant e.g., a RT functional fragment
  • 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 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 MMLV RT set forth in SEQ ID NO: 855), a RT set forth in SEQ ID NO: 856, or a RT domain, for example, provided in Tables 1, 3, 7, and 10.
  • a reference RT e.g., a MMLV RT set forth in SEQ ID NO: 855
  • the reference RT is a M-MLV RT set forth in SEQ ID NO: 855.
  • 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 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 M-MLV RT set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or a RT domain, for example, provided in Tables 1, 2, 3,
  • the reference RT is a M-MLV RT sequence set forth in SEQ ID NO: 855.
  • the RT truncated variant has a truncation at the N-terminal and the C-terminal end compared to a reference RT, e.g., a M-MLV-RT of SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or a RT domain, for example, provided in Tables 1, 2, 3, 4, 7, and 10.
  • the N-terminal truncation and the C-terminal truncation are of the same length.
  • the prime editors may include a functional variant of a reference M- MLV RT (e.g., as set forth in SEQ ID NO: 855).
  • the prime editors comprises an RT domain provided in Tables 1, 2, 3, 4, 7, and 10.
  • the RT or RT domain is a functional variant of a reference M-MLV RT (e.g., as set forth in SEQ ID NO: 855), a RT set forth in SEQ ID NO: 856, or a RT domain provided in Tables 1, 2, 3, 4, 7, and 10.
  • the functional variant of M-MLV RT is truncated after amino acid position 502 compared to a M- MLV RT as set forth in SEQ ID NO: 855, a RT set forth in SEQ ID NO: 856, or a RT domain provided in Tables 1, 23, 4, 7, and 10.
  • 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 as set forth in SEQ ID NO: 855, or a RT domain provided in Tables 1, 2, 3, 7, and 10, wherein X is any amino acid other than the original amino acid.
  • the WSGR Docket No.59761-781.601 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 as set forth in SEQ ID NO: 855, or a RT domain provided in Tables 1, 2, 3, 7, and 10 wherein X is any amino acid other than the original amino acid.
  • a DNA sequence encoding a prime editor comprising this truncated RT is 522 bp smaller than PE2, and therefore makes its potentially useful for applications where delivery of the DNA sequence is challenging due to its size (i.e., adeno-associated virus and lentivirus delivery).
  • the M-MLV RT variant consists of SEQ ID NO: 884.
  • the reverse transcriptase domain comprises an amino acid sequence that is at least 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% identical to any one of the sequences set forth in SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 130, 131, 204, 230, 232-244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 489, 990-1006, 209, 210, 231, or 229 provided in Tables 1, 2, 3, 7 and 10.
  • the reverse transcriptase domain comprises an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 5, 6, 13, 15, 16, 17, 18, 21, 22, 130, 131, 204, 230, 232-244, 249-257, 261, 270, 271, 327, 329, 332, 333, 337, 340, 341, 342, 344, 489, 990-1006, 209, 210, 231, or 229.
  • Exemplary reverse transcriptase domains are shown in Tables 1, 2, 3, 7, and 10.
  • the RT domain comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence selected from the group consisting of: SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, or 990-1006.
  • the RT domain comprises an amino acid sequence that is selected from the group consisting of: SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, or 990-1006.
  • the RT 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., deletions or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, or 990-1006.
  • RT domains used in prime editors can comprise multiple functional domains.
  • an RT domain may comprise a domain 1, a domain 2, a domain 3, a domain 4, a domain 5, a domain 6, a domain 7, and/or a Thumb domain.
  • a first polypeptide may comprise a domain 1, a domain 2, a domain 3, a domain 4, a domain 5, a domain 6, a domain 7, or a Thumb domain.
  • a domain 1, a domain 2, a domain 3, a domain 4, a domain 5, a domain 6, a domain 7, or a Thumb domain may also be part of a DNA polymerase domain, e.g., an RNA-mediated DNA polymerase domain.
  • a plurality of RT WSGR Docket No.59761-781.601 domains may share the domain structure of domain 1, domain 2, domain 3, domain 4, domain 5, domain 6, domain 7, and the Thumb domain.
  • the plurality of RT domains may be grouped into a plurality of RT families based on a specific sequence or structure feature in any of the domains thereof.
  • a plurality of RT domains may be grouped into six families as described in FIG.3.
  • a DNA polymerase domain in a prime editor may be modified compared to a wild type form.
  • a prime editor may comprise a truncated RT domain.
  • one or more domains of a naturally occurring RT is truncated or reduced for use in a prime editor.
  • the RT is a retroviral RT (e.g., MMLV-RT) wherein a RNaseH domain of the wild type retroviral RT is truncated or deleted.
  • amino acid sequences connecting one or more of domain 1 and domain 2, domain 2 and domain 3, domain 3 and domain 4, domain 4 and domain 5, domain 5 and domain 6, domain 6 and domain 7, or domain 7 and thumb domain of a naturally occurring RT may be truncated or deleted for use in a prime editor.
  • an RT domain of a prime editor may be selected from the group consisting of an nLTR RT domain, an LTR RT domain, a Group II intron RT domain, a Retron RT domain, a TERT RT domain, and an RVT_like RT domain.
  • an RT domain may be selected from the group consisting of a nLTR RT domain, an LTR RT domain, a Group II intron RT domain, a Retron RT domain, a TERT RT domain, and an RVT_like RT domain.
  • an RT domain of a prime editor may comprise an nLTR RT domain.
  • an RT domain of a prime editor may comprise an LTR RT domain. In some embodiments, an RT domain of a prime editor may comprise a Group II intron RT domain. In some embodiments, an RT domain of a prime editor may comprise a Retron RT domain. In some embodiments, an RT domain of a prime editor may comprise a TERT RT domain. In some embodiments, an RT domain of a prime editor may comprise an RVT_like RT domain. In some embodiments, a DNA polymerase domain or an RNA-mediated DNA polymerase domain of a prime editor may comprise the RT domain thereof or any combinations described herein. [0181] In some embodiments, a prime editor comprises an RT domain comprising an aspartic acid in domain 3.
  • a prime editor comprises an RT domain comprising the amino acid sequence YxDD in domain 5, wherein x is any amino acid.
  • a prime editor comprises an RT domain comprising an aspartic acid in domain 3 and the amino acid sequence YxDD in domain 5, wherein x is any amino acid.
  • the RT domain is a nLTR RT domain.
  • An nLTR RT domain of a prime editor may comprise any combinations of the amino acid or sequence described herein.
  • the amino acid or sequence described herein may also apply to an nLTR RT domain of an RT or a polypeptide.
  • the amino acid or sequence described herein may not be restricted to the nLTR RT domain of a prime editor.
  • a prime editor comprises an RT domain comprising the amino acid sequence PPxxxxIPK (SEQ ID NO: 905) in domain 1, wherein x is any amino acid.
  • a prime editor comprises an RT domain comprising the amino acid sequence QAIL (SEQ ID NO: 906) at position between domain 2 and domain 3.
  • a prime editor comprises an RT domain comprising the amino acid sequence RxLGIPxxDR (SEQ ID NO: 907) in domain 3, wherein x is any amino acid.
  • the prime editor comprises an RT domain comprising the amino acid sequence GTQGG (SEQ ID NO: 908) in domain 4.
  • the prime editor comprises an RT domain comprising the amino acid sequence ELERR (SEQ ID NO: 909) between domain 4 and domain 5. In some embodiments, the prime editor comprises an RT domain comprising the amino acid sequence LG in domain 7. In some embodiments, a prime editor comprises an RT domain comprising the amino acid sequence PPxxxxIPK (SEQ ID NO: 905) in domain 1, the amino acid sequence QAIL (SEQ ID NO: 906) at position between domain 2 and domain 3.
  • the amino acid sequence RxLGIPxxDR (SEQ ID NO: 907) in domain 3, the amino acid sequence GTQGG (SEQ ID NO: 908) in domain 4, the amino acid sequence ELERR (SEQ ID NO: 909) between domain 4 and domain 5, and/or the amino acid sequence LG in domain 7, or any combination thereof, where x is any amino acid.
  • the RT domain is a Group II intron RT domain.
  • a Group II intron RT domain of a prime editor may comprise any combinations of the amino acid or sequence described herein.
  • the amino acid or sequence described herein may also apply to a Group II intron RT domain of an RT or a polypeptide.
  • a prime editor may comprise a RT domain comprising the amino acid sequence NAxxH between domain 2 and domain 3, wherein x is any amino acid.
  • the prime editor comprises the amino acid sequence DFF in domain 3; GxxS in domain 4, wherein x is any amino acid; and/or YTRxxYxxDDxxS in domain 5, wherein x is any amino acid.
  • the prime editor comprises a RT domain comprising the amino acid sequence NAxxH (SEQ ID NO: 910) between domain 2 and domain 3, wherein x is any amino acid.
  • the prime editor comprises a RT domain comprising the amino acid sequence DFF in domain 3; or GxxS in domain 4.
  • the prime editor comprises a RT domain comprising the amino acid sequence YTRxxYxxDDxxS (SEQ ID NO: 910) in domain 5, wherein x is any amino acid.
  • the prime editor comprises a RT domain comprising the amino acid sequence NAxxH between domain 2 and domain 3; DFF at position in domain 3; GxxS in domain 4, and/or YTRxxYxxDDxxS (SEQ ID NO: 910) in domain 5, wherein x is any amino acid.
  • the RT domain is a Retron RT domain of a prime editor may comprise any combinations of the amino acid or sequence described herein.
  • the amino acid or sequence described herein may not be restricted to the Retron RT domain of a prime editor.
  • a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT.
  • the RT or RT domain is an eukaryotic RT, WSGR Docket No.59761-781.601 for example, a yeast, drosophila, rodent, or primate RT.
  • the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (GsI- IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT.
  • the RT or RT domain is a Group II intron RT, for example, a.
  • the prime editor comprises a retron RT.
  • RT or RT domain comprises a retron RT.
  • Ancestral Reverse transcriptase Components of prime editors described herein may comprise engineered protein sequence that share evolutionary ancestors with currently known proteins.
  • a prime editor may comprise a DNA polymerase that is reverse transcriptase (RT) polypeptide that comprises an ancestral sequence of a family of RTs.
  • a DNA polymerase domain of a prime editor comprises an engineered RT generated by ancestral sequence reconstruction, or ASR (such RTs referred to as ASR RTs).
  • ASR engineered RT generated by ancestral sequence reconstruction
  • Sequences from National Center for Biotechnology Information (NCBI), UniProt, EMBL, International Nucleotide Sequence Database Collaboration (INSDC), European Nucleotide Archive, or other databases may be used to construct ancestral sequences.
  • the collected sequences may be aligned by a multiple sequence alignment (MSA) algorithm.
  • MSA alignment algorithm may ClustalW, Kalign, MAFFT, MUSCLE, T-Coffee, derivatives thereof, or any combinations thereof.
  • Methods to handle gaps in sequence alignments may comprise Probabilistic Alignment Kit (PRANK) or any derivatives thereof.
  • an evolutionary model may be used to construct an ancestral phylogeny tree.
  • An evolutionary model may comprise Dayhoff models, for example, PAM120, PAM160, PAM250, or any derivatives thereof.
  • An evolutionary model may also comprise the JTT model, the WAG model, the LG model, the R10 model, the INV model, or the Blosum models.
  • a Blosum model may comprise Blosum45, Blosum62, Blosum80, or any derivatives thereof.
  • an evolutionary model may comprise computational constraints on the structure or function of the sequences. The constraints may be imposed by a computational model.
  • the fitness of an evolutionary model may also be evaluated using the Aikake Information Criterion or the Bayesian Information Criterion.
  • a phylogenetic tree may be constructed once the evolutionary model and its fitness are calculated.
  • a phylogenetic tree may comprise maximum likelihood methods.
  • a maximum likelihood method may comprise PhyML, MOLPHY, BioNJ, PHYLIP, or any derivatives thereof.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to any one of sequences selected from the WSGR Docket No.59761-781.601 group consisting of: SEQ ID NOs: 81-95.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to any one of sequences selected from the group consisting of: SEQ ID NOs: 81, 82, 84, 91, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, and 1105-1109.
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 81-95 or 1059-1109.
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 81, 82, 84, 91, or 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, or 1105-1109.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 81-95.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 81, 82, 84, 91, 1063, 1068, 1073, 1096, 1097, 1098, 1099, 1101, 1103, and 1105-1109.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences selected from Tables 1, 15, 16, 17, 18, or 19.
  • the RT domain comprises an WSGR Docket No.59761-781.601 amino acid sequence that is selected from any one of sequences selected from Tables 1, 15, 16, 17, 18, or 19.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1048-1089, 1090-1109, 1110-1124, 1125-1215, 1217-1291, 1292-1326, or 1327-1395.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 1048-1089, 1090-1109, 1110-1124, 1125-1215, 1217- 1291, 1292-1326, or 1327-1395.
  • the amino acid differences e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions do not include a difference at a conserved catalytic residue as shown in Table 23.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the WSGR Docket No.59761-781.601 amino acid sequences set forth in SEQ ID NOs: 1267, or 1288-1291.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 1267, or 1288-1291.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%,
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1308, 1312, 1315, 1316, 1325, or 1326.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 1308, 1312, 1315, 1316, 1325, or 1326.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%,
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, WSGR Docket No.59761-781.601 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1105 or 1367.
  • the RT domain comprises an amino acid sequence that is selected from any one of sequences set forth in SEQ ID NOs: 1105 or 1367.
  • an RT domain (e.g., an engineered RT) comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%,
  • an RT domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1177.
  • the RT domain comprises an amino acid sequence set forth in SEQ ID NO: 1177.
  • the amino acid differences, e.g., amino acid deletions, amino acid substitutions, or amino acid substitutions do not include a difference at a conserved catalytic residue as shown in Table 23.
  • a method of reverse transcribing a target RNA sequence may comprise contacting a target RNA sequence with an RT domain described herein.
  • the RT domain may reverse transcribe the RNA molecule into a complementary DNA sequence.
  • a cell may comprise the RT domains described herein.
  • Prime Editors with Solubility Enhancement (SET) domains [0194]
  • a prime editor described herein may comprise additional functional domains, for example, one or more domains that modify the folding, solubility, or charge of the prime editor.
  • the prime editor may comprise a SET domain.
  • the prime editor may comprise a SET domain from Table 5.
  • a SET domain may be associated, linked, or fused to any component of a prime editor (e.g., to a DNA polymerase domain and/or a DNA binding domain).
  • a SET domain is linked to a DNA-binding domain of a prime editor.
  • a SET domain is linked WSGR Docket No.59761-781.601 to a DNA polymerase domain of a prime editor.
  • the prime editor is a fusion protein
  • the SET domain may be positioned at the N-terminus of the prime editor, the C- terminus of the prime editor, or in between a DNA binding domain and a polymerase domain.
  • a SET domain may increase the solubility of a prime editor in vitro, relative to a prime editor without the SET domain.
  • the SET domain may increase the solubility of a prime editor in vivo, relative to a prime editor without the SET domain.
  • the increase in solubility of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, at least about 100 %, at least about 105 %, at least about 110 %, at least about 115 %, at least about 120 %, at least about 125 %, at least about 130 %, at least about 135 %, at least about 140 %, at least about 145 %, at least about 150 %, at least about 155 %, at least about
  • the increase in solubility of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be from 10 to 20 %, from 15 to 25 %, from 20 to 30 %, from 25 to 35 %, from 30 to 40 %, from 35 to 45 %, from 40 to 50 %, from 45 to 55 %, from 50 to 60 %, from 55 to 65 %, from 60 to 70 %, from 65 to 75 %, from 70 to 80 %, from 75 to 85 %, from 80 to 90 %, from 85 to 95 %, from 90 to 100 %, from 95 to 105 %, from 100 to 110 %, from 105 to 115 %, from 110 to 120 %, from 115 to 125 %, from 120 to 130 %, from 125 to 135 %, from 130 to 140 %, from 135 to 145 %, from 140 to 150 %, from 145 to 155 %, from 150 to 160 %, from 155 to 165
  • the increase in solubility of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be at least about at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8- fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35- fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.
  • the SET domain may increase the expression level of a prime editor in vitro, relative to a prime editor without the SET domain. In some embodiments, the SET domain may increase the expression level of a prime editor in vivo, relative to a prime editor without the SET domain.
  • the increase in expression level of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be at least about 10 %, at least WSGR Docket No.59761-781.601 about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, at least about 100 %, at least about 105 %, at least about 110 %, at least about 115 %, at least about 120 %, at least about 125 %, at least about 130 %, at least about 135 %, at least about 140 %, at least about 145 %, at least about 150
  • the increase in expression level of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be from 10 to 20 %, from 15 to 25 %, from 20 to 30 %, from 25 to 35 %, from 30 to 40 %, from 35 to 45 %, from 40 to 50 %, from 45 to 55 %, from 50 to 60 %, from 55 to 65 %, from 60 to 70 %, from 65 to 75 %, from 70 to 80 %, from 75 to 85 %, from 80 to 90 %, from 85 to 95 %, from 90 to 100 %, from 95 to 105 %, from 100 to 110 %, from 105 to 115 %, from 110 to 120 %, from 115 to 125 %, from 120 to 130 %, from 125 to 135 %, from 130 to 140 %, from 135 to 145 %, from 140 to 150 %, from 145 to 155 %, from 150 to 160 %, from 155 to 165
  • the increase in expression level of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be at least about at least about 1- fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5- fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.
  • the increase in expression level of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be from 1-fold to 2-fold, from 1.5-fold to 2.5-fold, from 2-fold to 3-fold, from 2.5-fold to 3.5-fold, from 3-fold to 4-fold, from 3.5-fold to 4.5-fold, from 4-fold to 5-fold, from 4.5-fold to 5.5-fold, from 5-fold to 6-fold, from 5.5-fold to 6.5-fold, from 6-fold to 7-fold, from 6.5-fold to 7.5-fold, from 7-fold to 8-fold, from 7.5-fold to 8.5-fold, from 8-fold to 9- fold, from 8.5-fold to 9.5-fold, from 9-fold to 10-fold, from 9.5-fold to 20-fold, from 15-fold to 25- fold, from 20-fold to 30-fold, from 25-fold to 35-fold, from 30-fold to 40-fold, from 35-fold to 45- fold, or from 40-fold to 50-fold.
  • a prime editor comprising the SET domain may increase prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity relative to a prime editor without the SET domain.
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor WSGR Docket No.59761-781.601 comprising the SET domain relative to a prime editor without the SET domain may be at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be from 10 to 20 %, from 15 to 25 %, from 20 to 30 %, from 25 to 35 %, from 30 to 40 %, from 35 to 45 %, from 40 to 50 %, from 45 to 55 %, from 50 to 60 %, from 55 to 65 %, from 60 to 70 %, from 65 to 75 %, from 70 to 80 %, from 75 to 85 %, from 80 to 90 %, from 85 to 95 %, from 90 to 100 %, from 95 to 105 %, from 100 to 110 %, from 105 to 115 %, from 110 to 120 %, from 115 to 125 %, from 120 to 130 %, from 125 to 135 %, from 130 to 140 %, from 135 to 145 %, from 140 to 150
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be at least about at least about 1- fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5- fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor comprising the SET domain relative to a prime editor without the SET domain may be from 1-fold to 2-fold, from 1.5-fold to 2.5-fold, from 2- fold to 3-fold, from 2.5-fold to 3.5-fold, from 3-fold to 4-fold, from 3.5-fold to 4.5-fold, from 4-fold to 5-fold, from 4.5-fold to 5.5-fold, from 5-fold to 6-fold, from 5.5-fold to 6.5-fold, from 6-fold to 7- fold, from 6.5-fold to 7.5-fold, from 7-fold to 8-fold, from 7.5-fold to 8.5-fold, from 8-fold to 9-fold, from 8.5-fold to 9.5-fold, from 9-fold to 10-fold, from 9.5-fold to 20-fold, from 15-fold to 25-fold, from 20-fold to 30-fold, from 25-fold to 35-fold, from 30-fold to 40-
  • a SET domain may adopt a secondary, tertiary, or quaternary structure when not fused to other components of the prime editor.
  • the SET domain may adopt a secondary structure without the prime editor.
  • the SET domain of a prime editor may adopt a tertiary structure without the prime editor.
  • the SET domain of a prime editor may adopt a quaternary structure without the prime editor.
  • the SET domain of a prime editor adopting a secondary, tertiary, or quaternary structure without the prime editor may comprise any size described herein.
  • the SET domain the SET domain of a prime editor may be less than about 100 kDa (kilo Dalton) or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 100 kDa. In some embodiments, the SET domain of a prime editor may be less than about 100 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 50 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor.
  • the SET domain of a prime editor may be less than about 50 kDa. In some embodiments, the SET domain of a prime editor may be less than about 50 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. [0203] In some embodiments, the SET domain of a prime editor may be less than about 20 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 20 kDa.
  • the SET domain of a prime editor may be less than about 20 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 10 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 10 kDa. In some embodiments, the SET domain of a prime editor may be less than about 10 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor.
  • the SET domain of a prime editor may be less than about 9 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 9 kDa. In some embodiments, the SET domain of a prime editor may be less than about 9 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 8 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor.
  • the SET domain of a prime editor may be less than about 8 kDa. In some embodiments, the SET domain of a prime editor may be less than about 8 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. [0204] In some embodiments, the SET domain of a prime editor may be less than about 7 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some WSGR Docket No.59761-781.601 embodiments, the SET domain of a prime editor may be less than about 7 kDa.
  • the SET domain of a prime editor may be less than about 7 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. [0205] In some embodiments, the SET domain of a prime editor may be less than about 10 kDa, 9 kDa, 8 kDa, 7 kDA or 6 kDa, or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 10 kDa, 9 kDa, 8 kDa, 7 kDA or 6 kDa.
  • the SET domain of a prime editor may be less than about 10 kDa, 9 kDa, 8 kDa, 7 kDA or 6 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 5 kDa or may adopt a secondary, tertiary, or quaternary structure without the prime editor. In some embodiments, the SET domain of a prime editor may be less than about 5 kDa. In some embodiments, the SET domain of a prime editor may be less than about 5 kDa and adopt a secondary, tertiary, or quaternary structure without the prime editor.
  • the SET domain of a prime editor may comprise a GB1 domain, a protein D domain, a Z domain of Staphylococcal protein A, a Fh8 domain, an MBP domain, a NusA domain, a Trx domain, a SUMO domain, a GST domain, a GB1 domain, a ZZ domain, a HaloTag domain, a SNUT domain, a Skp domain, a T7PK domain, an EspA domain, a Mocr domain, an Ecotin domain, a CaBP domain, an ArsC domain, an IF2-domain I domain, a RpoA domain, a SlyD domain, a Tsf domain, a RpoS domain, a PotD domain, a Crr domain, a msyB domain, an yjgD domain, a rpoD domain, a GFP domain, or a AK-tag domain.
  • the SET domain of a prime editor may comprise a protein D domain. In some embodiments, the SET domain of a prime editor may comprise a Z domain of Staphylococcal protein A. In some embodiments, the SET domain of a prime editor may comprise a Fh8 domain. In some embodiments, the SET domain of a prime editor may comprise an MBP domain. In some embodiments, the SET domain of a prime editor may comprise a NusA domain. In some embodiments, the SET domain of a prime editor may comprise, a Trx domain. In some embodiments, the SET domain of a prime editor may comprise a SUMO domain. In some embodiments, the SET domain of a prime editor may comprise a GST domain.
  • the SET domain of a prime editor may comprise a GB1 domain. In some embodiments, the SET domain of a prime editor may comprise a ZZ domain. In some embodiments, the SET domain of a prime editor may comprise a HaloTag domain. In some embodiments, the SET domain of a prime editor may comprise a SNUT domain. In some embodiments, the SET domain of a prime editor may comprise a Skp domain. In some embodiments, the SET domain of a prime editor may comprise a T7PK domain. In some embodiments, the SET domain of a prime editor may comprise an EspA domain. In some embodiments, the SET domain of a prime editor may comprise a Mocr domain.
  • the SET domain of a prime editor may comprise an Ecotin domain. In some embodiments, the SET domain of a prime editor may comprise a CaBP domain. In some embodiments, the SET domain of a prime editor may comprise an ArsC domain. In some WSGR Docket No.59761-781.601 embodiments, the SET domain of a prime editor may comprise an IF2-domain I domain. In some embodiments, the SET domain of a prime editor may comprise a RpoA domain. In some embodiments, the SET domain of a prime editor may comprise a SlyD domain. In some embodiments, the SET domain of a prime editor may comprise a Tsf domain.
  • the SET domain of a prime editor may comprise a RpoS domain. In some embodiments, the SET domain of a prime editor may comprise a PotD domain. In some embodiments, the SET domain of a prime editor may comprise a Crr domain. In some embodiments, the SET domain of a prime editor may comprise a msyB domain. In some embodiments, the SET domain of a prime editor may comprise an yjgD domain. In some embodiments, the SET domain of a prime editor may comprise a rpoD domain. In some embodiments, the SET domain of a prime editor may comprise a GFP domain. In some embodiments, the SET domain of a prime editor may comprise an AK-tag domain.
  • a SET domain of a prime editor comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence selected from the group consisting of: SEQ ID NOs: 96-128 or 137.
  • a SET domain (e.g., of an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., deletions or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 96-128 or 137 or in Table 5.
  • a SET domain of a prime editor comprises an amino acid sequence that is selected from the group consisting of: SEQ ID NOs: 96-128 or 137 or in Table 5.
  • the SET domain comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 102 and SEQ ID NO: 137.
  • a SET domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., deletions or substitutions compared to any of the amino acid sequences set forth in SEQ ID NO: 102 and SEQ ID NO: 137.
  • a SET domain of a prime editor comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 102 and SEQ ID NO: 137.
  • the SET domain comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about WSGR Docket No.59761-781.601 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence set forth in SEQ ID NO: 102.
  • a SET domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., deletions or substitutions compared to an amino acid sequences set forth in SEQ ID NO: 102.
  • a SET domain of a prime editor comprises an amino acid sequence set forth at SEQ ID NO: 102.
  • the SET domain comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence set forth in SEQ ID NO: 137.
  • a SET domain (e.g., an engineered RT) 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, 20, 25, 30, 35, 40, 45, or 50 differences e.g., mutations e.g., deletions or substitutions compared to an amino acid sequences set forth in SEQ ID NO: 137.
  • a SET domain of a prime editor comprises an amino acid sequence set forth at SEQ ID NO: 137.
  • a SET domain may increase the solubility, the expression level, the prime editing efficiency, the DNA polymerase activity, the DNA-binding activity, or the DNA endonuclease activity of a prime editor, relative to a prime editor lacking the SET domain.
  • a SET domain may increase the solubility, the expression level, the prime editing efficiency, the DNA polymerase activity, the DNA-binding activity, or the DNA endonuclease activity of a prime editor, relative to a prime editor lacking the SET domain.
  • a SET domain may increase the solubility of a prime editor, relative to a prime editor lacking the SET domain.
  • a SET domain may increase the expression level of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the prime editing efficiency of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the DNA polymerase activity of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the DNA-binding activity of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the DNA endonuclease activity of a prime editor, relative to a prime editor lacking the SET domain.
  • a SET domain may increase the solubility of a prime editor in vitro, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the solubility of a prime editor in vivo, relative to a prime editor lacking the SET domain.
  • the WSGR Docket No.59761-781.601 increase in solubility of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain, in some embodiments, may be at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, at least about 100 %, at least about 105 %, at least about 110 %, at least about 115 %, at least about 120 %, at least about 125 %, at least about 130 %, at least about 135 %, at least about 140 %, at least about 145 %, at
  • the increase in solubility of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be from 10 to 20 %, from 15 to 25 %, from 20 to 30 %, from 25 to 35 %, from 30 to 40 %, from 35 to 45 %, from 40 to 50 %, from 45 to 55 %, from 50 to 60 %, from 55 to 65 %, from 60 to 70 %, from 65 to 75 %, from 70 to 80 %, from 75 to 85 %, from 80 to 90 %, from 85 to 95 %, from 90 to 100 %, from 95 to 105 %, from 100 to 110 %, from 105 to 115 %, from 110 to 120 %, from 115 to 125 %, from 120 to 130 %, from 125 to 135 %, from 130 to 140 %, from 135 to 145 %, from 140 to 150 %, from 145 to 155 %, from 150 to 160 %, from 155 to
  • the increase in solubility of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be at least about at least about 1- fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5- fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.
  • the increase in solubility of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be from 1-fold to 2-fold, from 1.5-fold to 2.5-fold, from 2-fold to 3-fold, from 2.5-fold to 3.5-fold, from 3-fold to 4-fold, from 3.5-fold to 4.5-fold, from 4-fold to 5- fold, from 4.5-fold to 5.5-fold, from 5-fold to 6-fold, from 5.5-fold to 6.5-fold, from 6-fold to 7-fold, from 6.5-fold to 7.5-fold, from 7-fold to 8-fold, from 7.5-fold to 8.5-fold, from 8-fold to 9-fold, from 8.5-fold to 9.5-fold, from 9-fold to 10-fold, from 9.5-fold to 20-fold, from 15-fold to 25-fold, from 20- fold to 30-fold, from 25-fold to 35-fold, from 30-fold to 40-fold, from 35-fold to 45-fold, or from 40- fold to 50-fold.
  • a SET domain may increase the expression level of a prime editor in vitro, relative to a prime editor lacking the SET domain. In some embodiments, a SET domain may increase the expression level of a prime editor in vivo, relative to a prime editor lacking the SET WSGR Docket No.59761-781.601 domain.
  • the increase in expression level of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, at least about 100 %, at least about 105 %, at least about 110 %, at least about 115 %, at least about 120 %, at least about 125 %, at least about 130 %, at least about 135 %, at least about 140 %, at least about 145 %, at least about 150 %, at least about 155 %, at least about 160
  • the increase in expression level of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be from 10 to 20 %, from 15 to 25 %, from 20 to 30 %, from 25 to 35 %, from 30 to 40 %, from 35 to 45 %, from 40 to 50 %, from 45 to 55 %, from 50 to 60 %, from 55 to 65 %, from 60 to 70 %, from 65 to 75 %, from 70 to 80 %, from 75 to 85 %, from 80 to 90 %, from 85 to 95 %, from 90 to 100 %, from 95 to 105 %, from 100 to 110 %, from 105 to 115 %, from 110 to 120 %, from 115 to 125 %, from 120 to 130 %, from 125 to 135 %, from 130 to 140 %, from 135 to 145 %, from 140 to 150 %, from 145 to 155 %, from 150 to 160 %, from 155 to 165
  • the increase in expression level of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be at least about at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.
  • the increase in expression level of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be from 1-fold to 2-fold, from 1.5-fold to 2.5- fold, from 2-fold to 3-fold, from 2.5-fold to 3.5-fold, from 3-fold to 4-fold, from 3.5-fold to 4.5-fold, from 4-fold to 5-fold, from 4.5-fold to 5.5-fold, from 5-fold to 6-fold, from 5.5-fold to 6.5-fold, from 6-fold to 7-fold, from 6.5-fold to 7.5-fold, from 7-fold to 8-fold, from 7.5-fold to 8.5-fold, from 8-fold to 9-fold, from 8.5-fold to 9.5-fold, from 9-fold to 10-fold, from 9.5-fold to 20-fold, from 15-fold to 25-fold, from 20-fold to 30-fold, from 25-fold to 35-fold, from 30-fold to 40-fold, from 35-fold to 45- fold, or from 40-fold to 50-fold.
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be at least about 10 %, at least about 15 %, at least WSGR Docket No.59761-781.601 about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, at least about 100 %, at least about 105 %, at least about 110 %, at least about 115 %, at least about 120 %, at least about 125 %, at least about 130
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA- binding activity, or DNA endonuclease activity of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be at least about at least about 1- fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5- fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.
  • the increase in the prime editing efficiency, DNA polymerase activity, DNA-binding activity, or DNA endonuclease activity of a prime editor comprising a SET domain relative to a prime editor lacking the SET domain may be from 1-fold to 2-fold, from 1.5-fold to 2.5-fold, from 2-fold to 3-fold, from 2.5-fold to 3.5-fold, from 3-fold to 4-fold, from 3.5-fold to 4.5-fold, from 4-fold to 5- fold, from 4.5-fold to 5.5-fold, from 5-fold to 6-fold, from 5.5-fold to 6.5-fold, from 6-fold to 7-fold, from 6.5-fold to 7.5-fold, from 7-fold to 8-fold, from 7.5-fold to 8.5-fold, from 8-fold to 9-fold, from 8.5-fold to 9.5-fold, from 9-fold to 10-fold, from 9.5-fold to 20-fold, from 15-fold to 25-fold, from 20- fold to 30-fold, from 25-fold to 35-fold, from 30-fold to 40
  • the SET domain of a prime editor comprises a GB1 domain. In some embodiments, the SET domain of a prime editor comprises a GB1 domain. In some embodiments, a WSGR Docket No.59761-781.601 GB1 domain may increase the solubility, the expression level, the prime editing efficiency, the DNA polymerase activity, the DNA-binding activity, or the DNA endonuclease activity of a prime editor, relative to a prime editor lacking the GB1 domain. In some embodiments, a GB1 domain may increase the solubility of a prime editor, relative to a prime editor lacking the SET domain.
  • a GB1 domain may increase the expression level of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a GB1 domain may increase the prime editing efficiency of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a GB1 domain may increase the DNA polymerase activity of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a GB1 domain may increase the DNA-binding activity of a prime editor, relative to a prime editor lacking the SET domain. In some embodiments, a GB1 domain may increase the DNA endonuclease activity of a prime editor, relative to a prime editor lacking the SET domain.
  • a GB1 domain of a prime editor comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 102 and SEQ ID NO: 137.
  • a GB1 domain may be a basic GB1 (bGB1) domain.
  • a bGB1 domain may increase the solubility of a prime editor in vitro, relative to a prime editor comprising a GB1 domain.
  • a bGB1 domain may increase the solubility of a prime editor in vivo, relative to a prime editor comprising a GB1 domain.
  • a GB1 domain of a prime editor comprises an amino acid sequence of SEQ ID NO: 137.
  • a bGB1 domain may comprise asparagine at position 22 of SEQ ID NO: 102, arginine at position 36 of SEQ ID NO: 102, or lysine at position 42 of SEQ ID NO: 102. In some embodiments, a bGB1 domain may comprise asparagine at position 22 of SEQ ID NO: 102. In some embodiments, a bGB1 domain may comprise arginine at position 36 of SEQ ID NO: 102. In some embodiments, a bGB1 domain may comprise lysine at position 42 of SEQ ID NO: 102.
  • a bGB1 domain may comprise asparagine at position 22 of SEQ ID NO: 102, arginine at position 36 of SEQ ID NO: 102, and lysine at position 42 of SEQ ID NO: 102.
  • a bGB1 domain may have an isoelectric point (pI) of about 8.
  • a bGB1 domain may have an isoelectric point (pI) of about 8.1
  • a bGB1 domain may have an isoelectric point (pI) of about 8.2.
  • a bGB1 domain may have an isoelectric point (pI) of about 8.3.
  • a bGB1 domain may have an isoelectric point (pI) of about 8.4. In some embodiments, a bGB1 domain may have an isoelectric point (pI) of about 8.5. In some embodiments, a bGB1 domain may have an isoelectric point (pI) of about 8.6. In some embodiments, a bGB1 domain may have an isoelectric point (pI) of about 8.7. In some embodiments, a bGB1 domain may have an isoelectric point (pI) of about 8.8. In some embodiments, a bGB1 domain may have an isoelectric point (pI) of about 8.9.
  • a bGB1 domain may have an isoelectric point (pI) of 8.67.
  • DNA Binding Domain WSGR Docket No.59761-781.601 [0214]
  • the prime editors provided herein comprises a polypeptide domain having DNA binding activity (e.g., a DNA binding domain).
  • the prime editors provided herein comprise a DNA binding domain comprising an amino acid sequence at least 85% identical (e.g., 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% identical, or 100% identical) to any one of the sequences set forth in SEQ ID NOs: 1017-1026 (Table 4).
  • 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 or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1017-1026.
  • the prime editors provided herein comprises a DNA binding domain comprising an amino acid sequence that does not have a N-terminus methionine.
  • the prime editors provided herein comprises a DNA binding domain comprising an amino acid sequence comprising a N-terminus methionine.
  • the amino acid sequence of a DNA binding domain may be N-terminally modified by one or more processing enzymes, e.g., by Methionine aminopeptidases (MAP).
  • MAP Methionine aminopeptidases
  • the prime editors provided herein comprise a DNA binding domain comprising an amino acid sequence at least 85% identical (e.g., 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% identical, or 100% identical) to any one of the sequences set forth in SEQ ID NOs: 1019 or 1022.
  • 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 or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1019 or 1022.
  • the DNA binding domain comprises a nuclease activity, for example, RNA-guided DNA endonuclease activity of a Cas polypeptide.
  • the DNA binding domain comprises a nuclease domain or nuclease activity.
  • DNA binding domain comprises a nickase, or a fully active nuclease.
  • the prime editor comprises a DNA binding domain that is an inactive nuclease.
  • the DNA-binding domain 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.
  • a guide polynucleotide e.g., a PEgRNA
  • the polypeptide domain comprises a DNA binding domain. In some embodiments, the polypeptide domain comprises a DNA nickase domain. In some embodiments, a prime editor comprises a DNA binding domain and a DNA nickase domain. In some embodiments, WSGR Docket No.59761-781.601 the DNA-binding domain and the DNA nickase domain may comprise the same amino acid sequence. In one case, the DNA-binding domain and the DNA nickase domain may comprise overlapping amino acids.
  • the DNA-binding domain and the DNA nickase domain may comprise non-overlapping amino acids, e.g., the DNA-binding domain and the DNA nickase domain may comprise two independent amino acid sequences.
  • a prime editor may comprise more than one DNA-binding domain.
  • a prime editor may comprise more than one DNA nickase domain.
  • the polypeptide domain comprises a DNA binding domain.
  • the polypeptide domain comprises a DNA endonuclease domain.
  • a prime editor comprises a DNA binding domain and a DNA endonuclease domain.
  • the DNA-binding domain and the DNA endonuclease domain may comprise the same amino acid sequence. In one case, the DNA-binding domain and the DNA endonuclease domain may comprise overlapping amino acids. In some embodiments, the DNA-binding domain and the DNA endonuclease domain may comprise non-overlapping amino acids, e.g., the DNA-binding domain and the DNA endonuclease domain may comprise two independent amino acid sequences. In some embodiments, a prime editor may comprise more than one DNA-binding domain. In some embodiments, a prime editor may comprise more than one DNA endonuclease domain.
  • a prime editor may comprise DNA-binding activity, a DNA nickase activity, or a DNA endonuclease activity. In some embodiments, a prime editor may comprise a DNA-binding activity, a DNA nickase activity or a DNA endonuclease activity. In some embodiments, a prime editor may comprise a DNA endonuclease activity or a DNA nickase activity. In some embodiments, a prime editor may comprise a DNA-binding activity and either a DNA endonuclease activity or a DNA nickase activity.
  • the DNA-binding domain of a prime editor may comprise a Cas protein.
  • the Cas protein is a type V Cas protein.
  • the Cas protein is a Cas12i2 protein.
  • a Cas12i2 protein may comprise one or more domains. Non-limiting examples of 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 Cas12i2 protein domain comprises a guide nucleic acid recognition and/or a binding domain that may interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.
  • a Cas12i2 protein may comprise a chimeric Cas12i2 protein that is fused to other proteins or polypeptides.
  • a Cas12i2 protein may comprise be a chimera of various Cas12i2 proteins, for example, comprising domains from different Cas12i2 proteins.
  • a Cas12i2 protein as used herein may be a wildtype or a modified form of a Cas12i2 protein.
  • a Cas12i2 protein can be an active variant, inactive variant, or fragment of a wild type or modified Cas12i2 protein.
  • a Cas12i2 protein as described herein may comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas12i2 protein.
  • a Cas12i2 protein may be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 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 Cas12i2 protein.
  • a Cas12i2 protein may be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to SEQ ID NOs: 1017-1021.
  • a Cas12i2 protein may be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to SEQ ID NOs: 1019 or 1022.
  • a Cas12i2 protein may comprise 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences (e.g., mutations e.g., deletions or substitutions) compared to a wild type exemplary Cas12i2 protein.
  • a Cas12i2 protein may comprise 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences (e.g., mutations e.g., deletions or substitutions) compared to SEQ ID NOs: 1017-1021.
  • a Cas12i2 protein may comprise 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences (e.g., mutations e.g., deletions or substitutions) compared to SEQ ID NOs: 1019 or 1022.
  • Variants or fragments may comprise at least about 5%, 10%, 20%, 30%, 40%, 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 or modified Cas12i2 protein or a portion thereof. Variants or fragments may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to SEQ ID NOs: 1017-1021.
  • Variants or fragments may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to SEQ ID NOs: 1019 or 1022. Variants or fragments can be targeted to a nucleic acid locus in complex with a guide nucleic acid while lacking nucleic acid cleavage activity.
  • a Cas12i2 protein may comprise one or more nuclease domains, such as DNase domains.
  • a Cas12i2 protein may comprise a RuvC-like nuclease domain.
  • the RuvC domain may cut a different strand of double- stranded DNA to make a double-stranded break in the DNA.
  • a Cas12i2 protein may comprise only one nuclease domain (e.g., Cas12i2 comprises RuvC domain but lacks an WSGR Docket No.59761-781.601 HNH domain).
  • a Cas12i2 protein may be modified to comprise additional nuclease domains, e.g., an HNH domain from another Cas protein or an additional RuvC domain from Cas12i2 or another Cas protein.
  • a Cas12i2 protein may comprise an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a RuvC nuclease domain of a wild-type Cas12i2 protein.
  • a Cas12i2 protein may comprise 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 differences e.g., mutations e.g., deletions or substitutions compared to a nuclease domain (e.g., RuvC domain) of a wild-type Cas12i2 protein.
  • a Cas12i2 protein may be modified to optimize regulation of gene expression.
  • a Cas12i2 protein may be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and/or enzymatic activity.
  • Cas12i2 proteins may also be modified to change any other activity or property of the protein, such as stability.
  • a nuclease domain of the Cas12i2 protein may be modified, deleted, or inactivated, or a Cas12i2 protein may 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 Cas12i2 protein for regulating gene expression.
  • a Cas12i2 protein may be a fusion protein.
  • a Cas12i2 protein may be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • a Cas12i2 protein may be fused to a recombinase protein.
  • a Cas12i2 protein may also be fused to a heterologous polypeptide providing increased or decreased stability.
  • the fused domain or heterologous polypeptide may be located at the N-terminus, the C- terminus, or internally within the Cas12i2 protein.
  • a Cas12i2 protein may be provided in any form.
  • a Cas12i2 protein may be provided in the form of a protein, such as a Cas12i2 protein alone or complexed with a guide nucleic acid.
  • a Cas12i2 protein may be provided in the form of a nucleic acid encoding the Cas12i2 protein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA.
  • the nucleic acid encoding the Cas12i2 protein may be codon optimized for efficient translation into protein in a particular cell or organism.
  • Nucleic acids encoding Cas12i2 proteins may be stably integrated in the genome of the cell.
  • Nucleic acids encoding Cas12i2 proteins may be operably linked to a promoter active in the cell.
  • Nucleic acids encoding Cas12i2 proteins may be operably linked to a promoter in an expression construct.
  • Expression constructs may include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a Cas12i2 gene) and which may transfer such a nucleic acid sequence of interest to a target cell.
  • the Cas12i2 molecule or Cas12i2 domain comprises a responsive intein.
  • a DNA binding domain may comprise a split Cas12i2.
  • a split refers to division into two or WSGR Docket No.59761-781.601 more fragments.
  • a split Cas12i2 protein may include an active nuclease, a nickase, and a nuclease-null Cas12i2 protein.
  • a split Cas12i2 reconstitutes a full-length Cas12i2 protein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, 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%, or at least 100% efficiency compared to a Cas12i2 that is not split.
  • a Cas12i2 protein may comprise a modified form of a wild type Cas12i2 protein.
  • the modified form of the wild type Cas12i2 protein may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the Cas12i2 protein.
  • the modified form of the Cas12i2 protein may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type Cas12i2 protein.
  • the modified form of Cas12i2 protein may have no substantial nucleic acid-cleaving activity.
  • a Cas12i2 protein When a Cas12i2 protein is a modified form that has no substantial nucleic acid-cleaving activity, it may be referred to as enzymatically inactive and/or “dead” (abbreviated by “d”).
  • dCas12i2 A dead Cas12i2 protein (dCas12i2) may bind to a target polynucleotide but may not cleave the target polynucleotide.
  • Enzymatically inactive can refer to a polypeptide that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner but may not cleave a target polynucleotide.
  • An enzymatically inactive site-directed polypeptide may comprise an enzymatically inactive domain (e.g. nuclease domain).
  • Enzymatically inactive can refer to no activity.
  • Enzymatically inactive may refer to substantially no activity.
  • Enzymatically inactive can refer to essentially no activity.
  • Enzymatically inactive can refer to an 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., nucleic acid cleaving activity, wild-type Cas12i2 activity).
  • a Cas12i2 protein may comprise one or more mutations relative to a wild-type version of the protein.
  • the mutation can result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type Cas12i2 protein.
  • the mutation may result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid.
  • the mutation may result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of the target nucleic acid.
  • the mutation may result in one or more of the plurality of nucleic acid-cleaving domains lacking the ability to cleave the complementary strand and the non-complementary strand of the target nucleic acid.
  • the residues to be mutated in a nuclease domain may correspond to one or more catalytic residues of the nuclease.
  • the DNA-binding domain comprises a Cas12i2 protein domain that is a nickase.
  • the Cas12i2 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 Cas12i2 nickase comprises an amino acid substitution in a RuvC domain. Sequences of exemplary Cas12i2 nickases are provided in Table 4.
  • a Cas12i2 protein domain provided herein can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas12i2 protein.
  • a Cas12i2 protein domain provided herein can comprise an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas12i2 protein.
  • a Cas12i2 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 an exemplary Cas12i2 protein domain provided herein.
  • a Cas12i2 protein domain may be a fusion protein.
  • a Cas12i2 protein domain provided herein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain.
  • a Cas12i2 domain protein can also be fused to a heterologous polypeptide providing increased or decreased stability.
  • the fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein domain.
  • the Cas12i2 protein domain recognizes the PAM sequence 5'-TBN-3', wherein N is any nucleotide and B is one or more of C, G, or T.
  • the Cas12i2 protein domain recognizes the PAM sequence 5'-TTTN-3’, wherein N is any nucleotide.
  • the Cas12i2 protein domain recognizes the PAM sequence 5' -TTN-3', wherein N is any nucleotide.
  • the Cas12i2 protein domain recognizes the PAM sequence 5'- GTTB-3', wherein B is C, G, or T. In some embodiments, the Cas12i2 protein domain recognizes a PAM sequence of 5'-GTTK-3', 5'-VTTK-3', 5'-VTTS-3', 5'-TTTS-3' or 5'-VTTN-3', where K is G or T, V is A, C or G, and S is C or G. In some embodiments, the Cas12i2 protein domain recognizes a PAM sequence of 5'-TTTA-3' or 5’-NTTN-3’, wherein N is any nucleotide.
  • a prime editor provided herein comprises a Cas12i2 protein domain that contains modifications that allow altered PAM recognition.
  • a prime editor comprises a DNA binding domain that has nickase activity to cleave a first strand of a double stranded target DNA sequence.
  • the prime editor may cleave a first stand of a double stranded target DNA sequence.
  • the first strand of a double stranded target DNA sequence cleavable a prime editor may comprise a PAM sequence.
  • a Cas12i2 protein domain comprises one or more nuclease domains.
  • a Cas12i2 protein domain may 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 of a wild-type Cas12i2 protein.
  • a Cas12i2 protein domain comprises a single nuclease domain.
  • a prime editor comprises a Cas12i2 protein domain 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 Cas12i2 nuclease activity).
  • exemplary Cas12i2 protein domain sequences are shown in Table 4.
  • the Cas12i2 protein domain is at least 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% identical to a sequence provided in Table 4.
  • the Cas12i2 protein domain comprises an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 1017-1021.
  • the Cas12i2 protein domain comprises an amino acid sequence identical to any one of the sequences set forth in SEQ ID NOs: 1019 or 1022.
  • a Cas12i2 protein may comprise a wildtype Cas12i2 protein or a variant Cas12i2 protein, functional portion of any of these, fusion protein of any of these, or any combinations thereof.
  • a Cas12i2 polypeptide may comprise a wildtype Cas12i2 polypeptide.
  • a Cas12i2 polypeptide may comprise a variant Cas12i2 polypeptide.
  • the Cas12i2 protein domain recognizes a PAM sequence flanked by a spacer. In some embodiments, the spacer is on the 5’ end of the PAM sequence. In some embodiments, the spacer is on the 3’ end of the PAM sequence.
  • the spacer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the DNA binding domain of a prime editor e.g., Cas12i2 domain
  • the second polypeptide of a prime editor i.e., the DNA binding domain of the prime editor
  • the second polypeptide of the prime editor may be configured to cleave the first strand of a double stranded target DNA sequence at a cleavage site 3’ of a PAM sequence.
  • the cleavage site is 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 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 1 nucleotide downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 2 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 3 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 4 nucleotides downstream or 3’ of the PAM sequence.
  • the cleavage site is at least 5 WSGR Docket No.59761-781.601 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 6 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 7 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 8 nucleotides downstream or 3’ of the PAM sequence. In some cases, the cleavage site is at least 9 nucleotides downstream or 3’ of the PAM sequence.
  • the cleavage site is from 10 to 20 nucleotides, from 15 to 25 nucleotides, from 20 to 30 nucleotides, from 25 to 35 nucleotides, from 30 to 40 nucleotides, from 35 to 45, or from 45 to 50 nucleotides downstream or 3’ of the PAM sequence.
  • the DNA binding domain of a prime editor e.g., Cas12i2 domain, may be configured to cleave the first strand of a double stranded target DNA sequence at a cleavage site upstream of a PAM sequence.
  • the second polypeptide of a prime editor may be configured to cleave the first strand of a double stranded target DNA sequence at a cleavage site 5’ of a PAM sequence.
  • the cleavage site is 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 nucleotides upstream or 5’ of the PAM sequence.
  • the cleavage site is at least 1 nucleotide upstream or 5’ of the PAM sequence.
  • the cleavage site is at least 2 nucleotides upstream or 5’ of the PAM sequence.
  • the cleavage site is at least 3 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is at least 4 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is at least 5 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is at least 6 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is at least 7 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is at least 8 nucleotides upstream or 5’ of the PAM sequence.
  • cleavage site is at least 9 nucleotides upstream or 5’ of the PAM sequence. In some cases, the cleavage site is from 10 to 20 nucleotides, from 15 to 25 nucleotides, from 20 to 30 nucleotides, from 25 to 35 nucleotides, from 30 to 40 nucleotides, from 35 to 45, or from 45 to 50 nucleotides upstream or 5’ of the PAM sequence.
  • a Cas protein domain provided herein comprises a Cas fragment that is a functional fragment of a Cas protein domain provided herein that retains one or more Cas activities. In some embodiments, the Cas fragment is at least 100 amino acids in length.
  • a Cas12i2 domain may comprise an amino acid sequence comprising at least 85% identical (e.g., 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% identical, or 100% identical) to any one of the sequences set forth in: SEQ ID NOs:1017- 1021.
  • the Cas12i2 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., WSGR Docket No.59761-781.601 deletions or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 1017-1021.
  • a prime editor comprises a Cas12i2 protein domain that comprises a circular permutant Cas12i2 variant.
  • a Cas12i2 protein domain of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas protein domain (e.g., a Cas protein domain provided herein) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA).
  • gRNA guide RNA
  • An exemplary circular permutant configuration may be N-terminus– [original C-terminus]–[original N-terminus]–C-terminus.
  • Any of the Cas12i2 proteins described herein, including any variant, ortholog, or naturally occurring Cas12i2 or equivalent thereof, may be reconfigured as a circular permutant variant.
  • the circular permutants of a Cas12i2 protein domain may have the following structure: N-terminus–[original C-terminus]–[optional linker]–[original N-terminus]–C- terminus.
  • the circular permutant can be formed by linking a C-terminal fragment of a Cas12i2 to an N-terminal fragment of a Cas12i2, either directly or by using a linker, such as an amino acid linker.
  • the C-terminal fragment may correspond to the C-terminal 95% or more of the amino acids of a Cas protein domain provided herein, or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas protein domain (e.g., as set forth Table 4).
  • 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 Cas protein domain provided herein (e.g., as set forth Table 4). In some embodiments, 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 Cas12i2 protein domain provided herein (e.g., as set forth Table 4).
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 410 residues or less of a Cas12i2 protein domain provided herein (e.g., as set forth Table 4).
  • 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 Cas12i2 protein domain provided herein WSGR Docket No.59761-781.601 (e.g., as set forth Table 4).
  • a prime editor may comprise a DNA binding domain and a DNA polymerase domain (e.g., 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.
  • a linker may comprise a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, identical to a sequence set forth in SEQ ID NOs: 273-318 or 1014-1016. In some embodiments, a linker may comprise a sequence set forth in any one of SEQ ID NOs: 272-318 or 1014-1016.
  • polypeptides comprising components of a prime editor may be provided in trans relevant to each other. For example, a reverse transcriptase domain provided herein may be expressed, delivered, or otherwise provided as an individual component rather than as a part of a fusion protein with the DNA binding domain.
  • 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.
  • 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 amino acid sequence of the MCP is: GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTI KVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIY (SEQ ID NO: 912).
  • components of a prime editor are directly fused to each other.
  • components of a prime editor are associated to each other via a linker.
  • 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.).
  • the prime editors provided herein comprise a linker between the Cas12i2 protein domain and the prime editing domain (e.g., reverse transcriptase domain).
  • the linker may affect the editing efficiency by the prime editor as well as the stability and half-life of the prime editor.
  • the linker may be between 10 and 200 amino acids in length, between 15 and 155 amino acids in length, or at least 30 and 300 amino acids in length.
  • the linker may be at least 10 amino acids in length, at least 20 amino acids in length, at least 30 amino acids in length, at least WSGR Docket No.59761-781.601 40 amino acids in length, or at least 50 amino acids in length.
  • the linker is about 15 amino acids in length. In some embodiments, the linker is about 25 amino acids in length. In some embodiments, the linker is about 50 amino acids in length. In some embodiments, the linker is about 100 amino acids in length. In some embodiments, the linker is about 155 amino acids in length.
  • a “natural linker” is a linker that has evolved by nature to maintain activity between protein domains (e.g., between Cas protein domain and reverse transcriptase domain).
  • the natural linker comprises at least 1 domain, at least 2 domains, at least 3 domains, at least 4 domains, at least 5 domains, at least 6 domains, at least 7 domains, at least 8 domains, at least 9 domains, or at least 10 domains.
  • An exemplary natural linker is shown in FIG.2.
  • a “structured linker” is a linker that maintains structural integrity and increases stability of protein domains (e.g., between Cas protein domain and reverse transcriptase domain).
  • the structured linker comprises a fixed distance between protein domains (e.g., 1 domain, 2 domains, 3 domains, 4 domains, 5 domains, 6 domains, 7 domains, 8 domains, 9 domains, or 10 domains.).
  • the fixed distance is at least 1 nucleotide, 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, or at least 10 nucleotides.
  • An exemplary structured linker is shown in FIG.2.
  • an “unstructured linker” is a linker that is structurally flexible and allows access to productive conformations of protein domains (e.g., between Cas protein domain and reverse transcriptase domain).
  • the unstructured linker comprises at least 1 domain, at least 2 domains, at least 3 domains, at least 4 domains, at least 5 domains, at least 6 domains, at least 7 domains, at least 8 domains, at least 9 domains, or at least 10 domains.
  • An exemplary unstructured linker is shown in FIG.2.
  • the linker of the disclosure may comprise a PE2 linker, a GS11 linker, a ALEA linker, a GSS linker, a NAT17 linker, a NAT 23 linker, or a GS8 linker.
  • the linker is at least 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% identical to a sequence provided in Table 8.
  • the linker comprises an amino acid sequence selected from SEQ ID NOs: 272-318 or 1014-1016 (Table 8). Exemplary linkers are shown in Table 8. [0265] A person of skill in the art would appreciate that the present disclosure is not limited by the sequences in Table 8 and structures in FIG.2 as the configurations in Table 8 and FIG.2 are examples of a broader class of linkers included in the present disclosure. [0266] In certain embodiments, 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, WSGR Docket No.59761-781.601 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 may be flexible, rigid, and/or cleavable. In some embodiments, the linker is a flexible linker.
  • the linker comprises the amino acid sequence SGGSSGGSSGS ETPGTSESATPESSGGSSGGS (SEQ ID NO: 921). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 922). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 923). In other embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 924). [0268] In some embodiments, a linker comprises 1-100 amino acids.
  • the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 920). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 921). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 922). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 923).
  • the linker comprises the amino acid sequence GGSGGS (SEQ ID NO: 925), GGSGGSGGS (SEQ ID NO: 926), or SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 924), or SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 316).
  • the amino acid linkers are homologous to the endogenous amino acids that exist between such domains in a native polypeptide. In some embodiments the endogenous amino acids that exist between such domains are substituted but the length is unchanged from the natural length.
  • two or more components of a prime editor are linked to each other by a non-peptide linker.
  • 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, etc.).
  • the linker WSGR Docket No.59761-781.601 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, etc.).
  • the linker 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.
  • 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]–[polymerase]–COOH; or NH2–[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.
  • 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.
  • a prime editor further comprises one or more nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • 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 NLS may be linked or fused to the C-terminus of a DNA binding domain.
  • a NLS may be linked or fused to the N-terminus of a DNA binding domain.
  • a NLS may be linked or fused to the C-terminus of a DNA polymerase domain.
  • a NLS may be linked or fused to the N-terminus of a DNA polymerase domain.
  • a first NLS may be linked or fused to the C- terminus of a DNA binding domain and a second NLS may be linked or fused to the N-terminus of a DNA binding domain.
  • a NLS may be linked or fused to the N-terminus of a DNA polymerase domain.
  • a first NLS may be linked or fused to the C- terminus of a DNA polymerase domain and a second NLS may be linked or fused to the N-terminus of a DNA polymerase domain.
  • a first NLS may be linked or fused to the C- terminus of a DNA binding domain and a second NLS may be linked or fused to the N-terminus of a DNA polymerase domain.
  • a first NLS may be linked or fused to the C- terminus of a DNA polymerase domain and a second NLS may be linked or fused to the N-terminus of a DNA binding domain.
  • the first and the second NLs are identical.
  • the first and the second NLS are different.
  • a prime editor or prime editing complex comprises at least one NLS.
  • a prime editor or prime editing complex comprises at least two NLSs.
  • the NLSs can be the same NLS, or they can be different NLSs.
  • a prime editor may further comprise at least one nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • a prime editor may further comprise 1 NLS.
  • a prime editor may further comprise 2 NLSs.
  • a prime editor may further comprise 3 NLSs.
  • a primer editor may further comprise more than 4, 5, 6, 7, 8, 9 or 10 NLSs.
  • the NLSs may be expressed as part of a prime editor complex.
  • 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 WSGR Docket No.59761-781.601 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 is fusion protein that comprises an NLS at the N-terminus. In some embodiments, a prime editor is fusion protein that comprises an NLS at the C terminus. In some embodiments, a prime editor is fusion protein that comprises at least 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. [0278] 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).
  • the one or more NLSs of a prime editor comprise bipartite NLSs.
  • 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.
  • a prime editor comprises an amino acid sequence comprising a nuclear localization signal (NLS) having the sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 524), KRTADGSEFESPKKKRKV (SEQ ID NO: 927), KRTADGSEFEPKKKRKV (SEQ ID NO: 928), NLS KRPAAIKKAGQAKKKK (SEQ ID NO: 929), RQRRNELKRSF (SEQ ID NO: 930), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 931).
  • NLS nuclear localization signal
  • a NLS is a SV40 large T antigen NLS PKKKRKV (SEQ ID NO: 522).
  • 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 amino acid sequence comprises the Xenopus nucleoplasmin sequence KRXXXXXXXXKKKL (SEQ ID NO: 1047) wherein X is any amino acid.
  • 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. [0280] Other non-limiting examples of NLS sequences are provided in Table 9 below. Engineered Cas-RT [0281] In some embodiments.
  • Prime editors described herein comprise an engineered Cas-RT fusion protein or protein complexes, or a DNA binding domain (e.g., a Cas protein) -DNA polymerase (e.g., RT) fusion proteins or complexes.
  • a prime editor may comprise a fusion protein of a Cas polypeptide and a RT polypeptide, where the Cas component of a naturally occurring fusion protein of a Cas-RT and a Type III Cas protein is replaced by a heterologous DNA binding domain, e.g., a WSGR Docket No.59761-781.601 heterologous Cas polypeptide.
  • a prime editor comprises a RT domain of a naturally occurring Cas-RT fusion protein, and a DNA binding domain that is heterologous Cas polypeptide, i.e., the Cas polypeptide that is different than the one in the corresponding naturally occurring Cas-RT fusion protein
  • a prime editor comprises a DNA binding domain and a RT domain that naturally occurs in a Type III CRISPR system Cas-RT fusion protein.
  • the Cas polypeptide in a naturally occurring Cas-RT fusion protein that is replaced by a heterologous Cas polypeptide comprises a Cas1 polypeptide, a Cas3 polypeptide, or a Cas6 polypeptide.
  • a prime editor comprises a DNA binding domain and an RT domain that naturally occurs in a Cas1-RT fusion protein (the RT also referred to as a “Cas1 RT”), and the DNA binding domain is not a Cas1 domain.
  • a prime editor comprises a DNA binding domain comprises a DNA binding domain and an RT domain that naturally occurs in a Cas6-RT fusion protein (the RT also referred to as a “Cas6 RT”), and the DNA binding domain is not a Cas6 domain.
  • a prime editor comprises a DNA binding domain comprises a DNA binding domain and an RT domain that naturally occurs in a Cas-RT fusion protein comprising Cas1 and Cas6 ((the RT also referred to as a “Cas1-Cas6 RT”), and the DNA binding domain is not a Cas1 or Cas6 domain.
  • the naturally occurring Cas-RT fusion may have a configuration of Cas1-RT- Cas6 or Cas6-RT-Cas1, where either of the Cas1 or Cas6 may be replaced by a heterologous DNA binding domain for the purpose of a prime editor.
  • the DNA binding domain- RT fusion or complex may further be able to interact with other CRISPR Cas proteins, e.g., Cas3.
  • heterologous means a non-native gene or protein component of, e.g., an engineered complex or fusion protein that does not naturally occur in the same organism, or in a naturally occurring fusion protein or complex, as other components of the complex or fusion protein, but which is engineered into the complex or fusion protein.
  • the DNA binding domain that is used to replace a Cas domain in a naturally occurring Cas- RT fusion can be any DNA binding domain as described herein.
  • the DNA binding domain comprises a Cas12i2 polypeptide, or any functional variant or fragment as described herein. In some embodiments, the DNA binding domain comprises nuclease activity, for example, a nickase activity. [0283] In some embodiments, the DNA binding domain comprises a Cas12i2 polypeptide or a Cas12i2 variant as described herein, or a functional fragment thereof. In some embodiments, the DNA binding domain comprises a Cas12i2 nickase as disclosed herein.
  • a RT domain of a Cas-RT comprises an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a sequence selected from the group consisting of: WSGR Docket No.59761-781.601 SEQ ID NOs: 129-136, 345, 368, 396, or 533-846.
  • a RT domain of a Cas-RT 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 or substitutions compared to any of the amino acid sequences set forth in SEQ ID NOs: 129-136 or 533-846.
  • the RT domain of a Cas-RT comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 129-136, 345, 368, 396, or 533-846.
  • the engineered Cas-RTs described herein may comprise additional functional domains, for example, any of the SET domains as described herein.
  • an engineered Cas-RT comprises a GB1 domain, or bGB1 domain described herein.
  • the engineered Cas-RT described herein and fragments thereof may also comprise any of the SET domain, GB1 domain, or bGB1 domain described herein.
  • the SET domain, GB1 domain, or bGB1 domain may comprise any SET domains, GB1 domains, or bGB1 domains described in this disclosure.
  • Domain structures of prime editors [0286]
  • a prime editor may comprise a fusion polypeptide comprising a first polypeptide and a second polypeptide.
  • a first polypeptide of a prime editor may be located at the N-terminus or C-terminus of the second polypeptide of the primer editor. In some embodiments, the first polypeptide is located at the N-terminus of the second polypeptide. In some embodiments, the first polypeptide is located at the C-terminus of the second polypeptide.
  • a prime editor may further comprise at least one nuclear localization sequence (NLS). In some embodiments, a prime editor may further comprise 1 NLS. In some embodiments, a prime editor may further comprise 2 NLSs. In other cases, a prime editor may further comprise 3 NLSs. In one case, a primer editor may further comprise more than 4, 5, 6, 7, 8, 9 or 10 NLSs.
  • the domain structure of a prime editor may comprise NH2-first polypeptide-second polypeptide-COOH or NH2-second polypeptide-first polypeptide-COOH, wherein NH2 is the N-terminus of the primer editor, wherein COOH is the C-terminus of the prime editor, wherein - comprises from 0-100 amino acids.
  • the domain structure of a prime editor may comprise NH2-first polypeptide-second polypeptide-COOH.
  • the domain structure of a prime editor may comprise NH2-second polypeptide-first polypeptide- COOH.
  • the domain structure of a prime editor comprising one NLS may comprise NH2-first polypeptide-second polypeptide-NLS-COOH, NH2-first polypeptide-NLS-second polypeptide-COOH, NH2-NLS-first polypeptide-second polypeptide-COOH, NH2-second polypeptide-first polypeptide-NLS-COOH, or NH2-NLS-second polypeptide-first polypeptide-COOH.
  • the domain structure of a prime editor comprising one NLS may comprise NH2-first polypeptide-second polypeptide-NLS-COOH.
  • the domain structure of a prime editor comprising WSGR Docket No.59761-781.601 one NLS may comprise NH2-first polypeptide-NLS-second polypeptide-COOH. In some embodiments, the domain structure of a prime editor comprising one NLS may comprise NH2-NLS- first polypeptide-second polypeptide-COOH. In some embodiments, the domain structure of a prime editor comprising one NLS may comprise NH2-second polypeptide-first polypeptide-NLS-COOH. In some embodiments, the domain structure of a prime editor comprising one NLS may comprise NH2- second polypeptide-NLS-first polypeptide-COOH.
  • the domain structure of a prime editor comprising one NLS may comprise NH2-NLS-second polypeptide-first polypeptide- COOH.
  • a prime editor comprising more than one NLS may have an NLS sequence located in the N-terminus or C-terminus of the first polypeptide or the second polypeptide.
  • an NLS sequence may be located in the N-terminus or C-terminus of another NLS.
  • a second polypeptide may comprise a DNA-binding domain or a DNA endonuclease domain.
  • a second polypeptide may comprise a DNA-binding domain.
  • the second polypeptide of a prime editor may comprise a DNA endonuclease domain.
  • a second polypeptide may comprise a DNA-binding domain and a DNA endonuclease domain.
  • the DNA-binding domain may comprise DNA endonuclease activity.
  • the DNA-binding domain and the DNA endonuclease domain may comprise overlapping amino acids.
  • the prime editor may comprise a DNA-binding domain and further a separate endonuclease domain.
  • the DNA-binding domain and the DNA endonuclease domain may comprise two independent amino acid sequences.
  • the second polypeptide of a prime editor may comprise more than one DNA-binding domain. In one case, the second polypeptide of a prime editor may comprise more than one DNA endonuclease domain.
  • the DNA-binding domain of a prime editor may be located at the N- terminus or C-terminus of the DNA endonuclease domain of the prime editor. In some embodiments, the DNA-binding domain is located at the N-terminus of the DNA endonuclease domain of the prime editor. In some embodiments, the DNA-binding domain of a prime editor is located at the C-terminus of the DNA endonuclease domain of the prime editor.
  • the DNA-binding domain of a prime editor is located within the DNA endonuclease domain of the prime editor. In other cases, the DNA endonuclease domain of a prime editor is located within the DNA-binding domain of the prime editor.
  • the DNA- binding domain may also be located at the N-terminus and the C-terminus of a DNA endonuclease domain.
  • the DNA endonuclease domain may also be located at the N-terminus and the C-terminus of a DNA-binding domain.
  • a prime editor may comprise the combinations of the arrangements of the DNA-binding domain and the DNA endonuclease domain described in this disclosure.
  • a prime editor further comprises additional polypeptide components, for example, a flap endonuclease (FEN, e.g., FEN1).
  • FEN flap endonuclease
  • the flap endonuclease excises the 5’ single stranded DNA of an edited strand, e.g., the 5’ single stranded DNA in the non- PAM strand formed at a nick generated by a Cas12i2 nuclease, of the target gene and assists incorporation of the intended nucleotide edit into the target gene.
  • the FEN is linked or fused to another component.
  • the FEN is provided in trans, for example, as a separate polypeptide or polynucleotide encoding the FEN.
  • a prime editor or prime editing composition comprises a flap nuclease.
  • the flap nuclease is a FEN1, or any FEN1 functional variant, functional mutant, or functional fragment thereof. In some embodiments, the flap nuclease is a TREX2, EXO1, or any other flap nuclease known in the art, or any functional variant, functional mutant, or functional fragment thereof. In some embodiments, the flap nuclease has 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 any of the flap nucleases described herein or known in the art.
  • a prime editor described herein may comprise additional functional domains, for example, one or more domains that modify the folding, solubility, or charge of the prime editor.
  • the prime editor may comprise a solubility-enhancement (SET) domain.
  • the prime editor may comprise a recombinase domain.
  • the prime editor may comprise a split intein.
  • the prime editor comprises an additional protein or protein domain that comprises a recombinase activity to effect site-specific recombination.
  • Site-specific recombination is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology.
  • SSRs site-specific recombinases
  • SSR recognition sequences DNA sequences
  • Site-specific recombination systems are highly specific, fast, and efficient, even when faced with complex eukaryotic genomes. Recombination sites are typically between about 30 and 200 nucleotides in length and generally consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place.
  • Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Tables 11, 12, 13 and 14 provide exemplary recombinases and their cognate SSR recognition sequences.
  • a prime editor comprises a serine recombinase.
  • the serine recombinase is a WSGR Docket No.59761-781.601 recombinase selected from Tables 11 and 12.
  • a prime editor comprises a tyrosine recombinase.
  • the tyrosine recombinase is selected from Tables 13 and 14.
  • installing one or more SSR recognition sequences may be achieved using single flap prime editing, dual prime editing, or multi-flap prime editing.
  • single flap, dual prime editing or multi-flap prime editing may be used to insert one or more or two or more SSR recognition sequences into a desired genomic site.
  • the recombinase is a serine recombinase.
  • the recombinase is a Bxb1 recombinase.
  • the recombinase is a phiC31 recombinase.
  • the recombinase is a serine recombinase as described herein, or any serine recombinase known in the art, or any functional variant thereof.
  • the recombinase recognition site introduced in the target DNA is an attP sequence
  • the second recombinase recognition site in the donor polynucleotide is an attB sequence.
  • the recombinase is a tyrosine recombinase.
  • the recombinase is a Cre recombinase.
  • the recombinase is a Flp recombinase.
  • the recombinase is a tyrosine recombinase disclosed herein, or any tyrosine recombinase known in the art.
  • the two or more recombinase recognition sites introduced in the target DNA each comprises a Lox sequence.
  • the two or more recombinase recognition sites introduced in the target DNA each individually comprises a different (orthogonal) Lox sequence, e.g., a LoxP sequence, a Lox511 sequence, a Lox66 sequence, a Lox71 sequence, or a Lox2272 sequence.
  • a prime editor system may insert one or more SSR recognition sequences into the double stranded DNA target.
  • a prime editor system may insert an SSR recognition sequence that is recognized by a cognate SSR that is provided by the prime editor system.
  • a prime editor system may insert an SSR recognition sequence that is recognized by a cognate SSR that is already present in a cell, tissue or organism.
  • a prime editor system may insert an SSR recognition sequence that is recognized by a cognate SSR that is separately introduced into a cell, tissue or organism.
  • the SSR recognition sequence is installed into a specific genomic site in a genome, i.e., into a double stranded DNA target sequence.
  • a single SSR recognition sequence is installed into a single specific genomic site. In some embodiments, a single SSR recognition sequence is installed into multiple specific genomic sites. In some embodiments, multiple SSR recognition sequences are installed within a genome. In some embodiments, multiple SSR recognition sequences are installed into multiple specific genomic sites. In some embodiments, two SSR recognition sequences are installed within a genome, e.g., into multiple specific genomic sites, wherein each SSR recognition sequence is recognized by a single cognate SSR. In some embodiments, a prime editor system comprising an SSR contacts a double stranded DNA target that already comprises one or more SSR recognition sequences.
  • a cognate SSR that recognizes an installed SSR recognition sequence is introduced to catalyze the precise cleavage, strand exchange, and rejoining of DNA fragments at the defined SSR recombination sites. This may be accomplished without relying on endogenous repair mechanisms in a cell for repairing double-strand breaks which otherwise can induce indels and other undesirable DNA rearrangements.
  • a reaction catalyzed by an SSR and one or more SSR recognition sequences results in large-scale genomic changes, such as, insertions, deletions, inversions, replacements, and chromosomal translocations of one or more chromosomal regions, including one or more loci, one or more genes, or one or more portions of genes (e.g., gene exons, introns, and gene regulatory regions).
  • the one or more SSR recognition sites can be inserted or introduced anywhere within genome. In some organisms, a genome is organized as a single chromosome (e.g., bacteria) and the SSR recognition site may be inserted at any locus within the chromosome.
  • the insertion site may be within a gene or within an intergenic region of a chromosome.
  • the insertion may be within an exon, intron, or therebetween, or within a regulatory sequence, such as a promoter, enhancer, or transcription binding sequence.
  • the genome is organized into more than one chromosome and the SSR recognition site may be inserted at any locus within the chromosome.
  • the genome also may be mitochondrial DNA.
  • the insertion site may be within a gene or within an intergenic region of a chromosome.
  • the insertion may be within an exon, intron, or within a regulatory sequence, such as a promoter, enhancer, or transcription binding sequence.
  • inserting in a genome in any organism can include inserting one or more SSR recognition sites in any one or more chromosomes of a given genome (depending upon the number of chromosomes making up the genome) and at any chromosomal locus or loci. Where a genome comprises more than one chromosome, reference to “inserting in a genome” may include inserting the one or more SSRs into the one or more chromosomes of the genome or into mitochondrial DNA.
  • a prime editor system comprising a recombinase can result in deletion of one or more nucleotides in a target DNA or target gene.
  • a prime editor system can result in integration of a first SSR recognition site and a second SSR recognition site in the target DNA, wherein the first and the second SSR recognition sites are in the same orientation, and wherein the recombinase mediates recombination between the two SSR recognition sites, thereby resulting in deletion of the sequence in between the first and the second SSR recognition sites.
  • a prime editor system comprising a recombinase can result in replacement of an endogenous sequence in a target DNA or a target gene by an exogenous DNA sequence.
  • a prime editor system can be used to insert a first SSR recognition site and a second SSR recognition site in the target DNA.
  • the prime editor system further comprises a donor DNA, wherein the donor DNA comprises a third and a forth SSR recognition sites, wherein the recombinase mediates recombination between the first SSR WSGR Docket No.59761-781.601 recognition site and the third SSR recognition site and recombination between the second SSR recognition site and the fourth SSR recognition site, thereby resulting in replacement of the sequence between the first and the second SSR recognition sites in the target DNA by the sequence between the third and the fourth SSR recognition sites in the donor DNA.
  • a prime editor comprises a split intein.
  • 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.
  • a split intein is derived from a eukaryotic intein, a bacterial intein, or an archaeal intein.
  • a split intein 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 substitutions that enhances the trans-splicing activity of the split intein.
  • the intein-C comprises 4 -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
  • 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).
  • GST glutathione- 5-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta- galactosidase beta-glucuronidase
  • luciferase green fluorescent protein
  • GFP green fluorescent protein
  • HcRed HcRed
  • DsRed cyan fluorescent protein
  • 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 comprises a protein domain that is capable of modifying the intracellular half-life of the prime editor.
  • components of a prime editors are arranged in a modular fashion to target, edit, or modify a target DNA sequence, e.g., to install a desired nucleotide edit into a target cell genome, by reverse transcription.
  • the components of a prime editor may comprise an unrelated DNA binding domain, and a DNA polymerase domain, e.g., reverse transcriptase domain.
  • the DNA binding domain and the DNA polymerase domain can be interchangeably located in the 5’ portion of the prime editor or the 3’ portion of the prime editor.
  • multiple functional domains may arise from a single protein. In some embodiments, all functional domains may arise from different proteins.
  • a DNA binding domain of a prime editor may be located C-terminal to the DNA polymerase domain. In some embodiments, a DNA binding domain of a prime editor may be located N-terminal to the DNA polymerase domain.
  • a prime editor may comprise a DNA polymerase domain having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855-857, 884, 990-1006, 1059-1109, 1048-1215, or 1217- 1395 and a DNA binding domain having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 1017-1021.
  • a prime editor may comprise a DNA polymerase domain having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 1-95, 129-136, 198-271, 319-493, 533-846, 855- 857, 884, 990-1006, 1059-1109, 1048-1215, or 1217-1395 and a DNA binding domain having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 1019 or 1022.
  • the prime editor further comprises a linker having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 272- 318, 913-926 or 1014.
  • a prime editor further comprises one or more nuclear localization sequence (NLS) having an amino acid sequence that is selected from any of the amino acid sequence selected from SEQ ID NOs: 522-532, 928-931, 1015 or 1016.
  • NLS nuclear localization sequence
  • the NLS is fused to the N-terminus of a DNA polymerase domain described herein.
  • the NLS is fused to the C-terminus of the DNA polymerase domain.
  • the NLS is fused to the N- terminus or the C-terminus of a DNA binding domain.
  • a linker sequence is disposed between the NLS and a domain of the prime editor.
  • PEgRNAs WSGR Docket No.59761-781.601 [0309]
  • the term “prime editing guide RNA”, or “PEgRNA”, refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA.
  • 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.
  • Nucleotide edit or “intended nucleotide edit” refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene.
  • Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence.
  • a single PEgRNA comprises a single nucleotide edit or intended nucleotide edit.
  • a single PEgRNA comprises multiple nucleotide edits or intended nucleotide edits.
  • a PEgRNA comprises at least one of: a spacer, an extension arm, and a gRNA core.
  • a PEgRNA comprises a spacer sequence 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 further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence can be referred to as an extension arm.
  • 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 a strand of the target gene at a nick site generated by the prime editor, for example, a free 3’ end at a nick site on the target strand generated by a Cas12i2 nuclease, or a free 3’ end at a nick site on the PAM strand generated by a Cas12i2 nuclease.
  • the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by prime editing.
  • 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 editing template comprises partial complementarity to an editing target sequence in the target gene.
  • the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene.
  • a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide.
  • a PEgRNA is a chimeric polynucleotide that includes both WSGR Docket No.59761-781.601 RNA and DNA nucleotides.
  • a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm.
  • a PEgRNA comprises DNA in the spacer sequence.
  • the entire spacer sequence 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.
  • a spacer may comprise a sequence that hybridizes to a first strand of a double stranded target DNA sequence.
  • the spacer may comprise complementary sequence to a search target sequence in the first strand of the double stranded DNA sequence.
  • an extension arm may comprise a sequence that hybridizes to a second strand (i.e., the complementary strand of the first strand) of the double stranded target DNA sequence.
  • a gRNA core may comprise a sequence that interacts with the second polypeptide of the prime editor (i.e., interacts with the DNA binding domain of the prime editor).
  • a nucleotide of a PEgRNA may be part of a spacer.
  • a nucleotide of a PEgRNA may be part of an extension arm.
  • a nucleotide of a PEgRNA may be part of a gRNA core.
  • a nucleotide of a PEgRNA may be part of a spacer and an extension arm.
  • a nucleotide of a PEgRNA may be part of a spacer and a gRNA core.
  • a nucleotide of a PEgRNA may be part of an extension arm and a gRNA core. In some embodiments, a nucleotide of a PEgRNA may be part of a spacer and an extension arm. In some embodiments, a nucleotide of a PEgRNA may not be part of a spacer, an extension arm, or a gRNA core. [0314] Components of a PEgRNA may be arranged in a modular fashion.
  • the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), 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 of the PEgRNA.
  • the gRNA core of a PEgRNA may be located at the 3’ end of a spacer.
  • 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, the PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, and an extension arm. In some embodiments, the PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS.
  • the WSGR Docket No.59761-781.601 PEgRNA comprises, from 5’ to 3’: an extension arm, a spacer, and a gRNA core.
  • the PEgRNA comprises, from 5’ to 3’: an editing target, a PBS, a spacer, and a gRNA core.
  • a PEgRNA comprises a gRNA core comprising a nucleotide sequence provided in Table 6.
  • a prime editor system comprising a Cas12i1 DNA binding domain comprises a PEgRNA comprising a gRNA core of SEQ ID NOs: 1037, 1038 or 1039.
  • a prime editor system comprising a Cas12i2 DNA binding domain comprises a PEgRNA comprising a gRNA core of SEQ ID NOs: 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035 or 1036.
  • a prime editor system comprising a Cas12i3 DNA binding domain comprises a PEgRNA comprising a gRNA core of SEQ ID NOs: 1040, 1041 or 1042.
  • a prime editor system comprising a Cas12i4 DNA binding domain comprises a PEgRNA comprising a gRNA core of SEQ ID NOs: 1043, 1044, 1045 or 1046.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-TBN-3', wherein N is any nucleotide and B is one or more of C, G, or T. In some embodiments, a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-TBN-3', wherein N is any nucleotide and B is one or more of C, G, or T. [0317] In some embodiments, a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5' -TTN-3', wherein N is any nucleotide.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5' -TTN- 3', wherein N is any nucleotide.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-GTTB-3', wherein B is C, G, or T.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-GTTB-3', wherein B is C, G, or T.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-GTTK-3', 5'-VTTK-3', 5'-VTTS-3', 5'-TTTS-3' or 5'-VTTN-3', where K is G or T, V is A, C or G, and S is C or G.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-GTTK-3', 5'-VTTK-3', 5'-VTTS-3', 5'- TTTS-3' or 5'-VTTN-3', where K is G or T, V is A, C or G, and S is C or G.
  • a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-TTTA-3' or 5’-NTTN-3’, wherein N is any nucleotide. In some embodiments, a PEgRNA of this disclosure is part of a prime editing system that recognizes the PAM motif 5'-TTTA-3' or 5’-NTTN-3’, wherein N is any nucleotide. [0321] In some embodiments, a PEgRNA comprises a single polynucleotide molecule that comprises the spacer sequence, the gRNA core, and the extension arm.
  • 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.
  • a spacer sequence comprises a region that has substantial complementarity to a search target sequence on the target strand of a double stranded target DNA.
  • the spacer sequence of a PEgRNA is identical or substantially identical to a protospacer sequence on the PAM strand of the target gene (except that the protospacer sequence comprises thymine and the spacer sequence may comprise uracil).
  • the spacer sequence is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a search target sequence in the target gene.
  • the spacer comprises is substantially complementary to the search target sequence.
  • a spacer may guide a prime editing complex to a genomic locus with identical sequence during prime editing.
  • 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 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides.
  • 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 12 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 17 to 23 nucleotides in length, 18 to 22 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 16-20 nucleotides in length. In some embodiments, the spacer is 17, 18, 19 or 20 nucleotides in length.
  • the spacer comprises a first spacer sequence comprising the 5’ half of the spacer and a second spacer sequence comprising the 3’ half of the spacer, wherein the tag sequence is between the first spacer sequence and the second spacer sequence.
  • the tag sequence does not have substantial complementarity to the spacer. In some embodiments, the tag does not have complementarity to the spacer.
  • a PEgRNA or a nick guide RNA 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 WSGR Docket No.59761-781.601 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.
  • the extension arm of a PEgRNA can comprise a primer binding site (PBS) and an editing template (e.g., an RTT).
  • the extension arm may be partially complementary to the spacer.
  • the editing template e.g., RTT
  • the editing template e.g., RTT
  • the primer binding site PBS
  • An extension arm of a PEgRNA can comprise a primer binding site sequence (PBS, or PBS sequence) that hybridizes with a free 3’ end of a single stranded DNA in the target 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 6 nucleotides in length. In some embodiments, the PBS is about 4 to 16 nucleotides, about 6 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 nucleotides in length. In some embodiments, 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.
  • the PBS is 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.
  • the PBS can be complementary or substantially complementary to a DNA sequence in a nicked strand of the target gene.
  • the PBS can be complementary or substantially complementary to a DNA sequence immediately upstream of a nick site on the target strand, wherein the nick site corresponds to a nick generated by a Cas12i2 nuclease complexed with the PEgRNA.
  • the PBS can initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site.
  • the PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the nicked PAM strand or target strand of the target gene.
  • the PBS is perfectly complementary, or 100% complementary, to a region of the nicked PAM strand or the target strand of the target 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.
  • WSGR Docket No.59761-781.601 The length of an editing template can 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 (RTT).
  • the editing template e.g., RTT
  • the editing template is 2-200 nucleotides in length. In some embodiments, the RTT is 5-200 nucleotides in length.
  • the RTT is 5-150 nucleotides in length, 5-125 nucleotides in length, 5-100 nucleotides in length, 5-75 nucleotides in length, 5-50 nucleotides in length, 5-45 nucleotides in length, 5-40 nucleotides in length, 5-35 nucleotides in length, 5-30 nucleotides in length, 5-25 nucleotides in length, 5-20 nucleotides in length, 10-150 nucleotides in length, 10-125 nucleotides in length, 10-100 nucleotides in length, 10- 75 nucleotides in length, 10-50 nucleotides in length, 10-45 nucleotides in length, 10-40 nucleotides in length, 10-35 nucleotides in length, 10-30 nucleotides in length, 10-25 nucleotides in length, 10-20 nucleotides, 15-150 nucleotides in length, 15-125 nucleotides in length, 15-100 nucleo
  • the 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 RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT 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. [0332] In some embodiments, the editing template (e.g., RTT) sequence is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the editing target sequence on a nicked strand of the target gene.
  • the RTT can be about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary a DNA sequence immediately downstream of a nick site on the target strand, wherein the nick site corresponds to a nick generated by a Cas12i2 nuclease complexed with the PEgRNA.
  • the editing template sequence e.g., RTT
  • the editing template sequence is complementary to the editing target sequence except at positions of the intended nucleotide edits to be incorporated int the target gene.
  • the editing template comprises a nucleotide sequence comprising about 85% to about 95% complementarity to an editing target sequence in the nicked PAM strand or target strand in the target gene. In some embodiments, the editing template comprises about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementarity to an editing target sequence in the nicked PAM strand or target strand of the target gene.
  • An intended nucleotide edit in an editing template of a PEgRNA may comprise various types of alterations as compared to the target gene sequence.
  • the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence.
  • the WSGR Docket No.59761-781.601 nucleotide edit is a deletion as compared to the target gene sequence.
  • the nucleotide edit is an insertion as compared to the target gene sequence.
  • the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence.
  • 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. In some embodiments, the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence.
  • a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution. In some embodiments, a nucleotide substitution comprises an A-to-guanine (G) substitution. In some embodiments, a nucleotide substitution comprises an A-to-cytosine (C) substitution. In some embodiments, a nucleotide substitution comprises a T-A substitution. In some embodiments, a nucleotide substitution comprises a T-G substitution. In some embodiments, a nucleotide substitution comprises a T-C substitution. In some embodiments, a nucleotide substitution comprises a G-to-A substitution.
  • 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 from 20-30 nucleotides, 25-40 nucleotides, 35- 50 nucleotides, 40-60 nucleotides, 50-100 nucleotides, 75-150 nucleotides, 125-250 nucleotides, 200- WSGR Docket No.59761-781.601 300 nucleotides, 250-400 nucleotides, 300-600 nucleotides, 500-750 nucleotides, 700-1000 nucleotides, or between 1000-2000 nucleotides in length.
  • a nucleotide insertion can be between 1 and 10 kb, 1 and 20 kb, 1 and 30 kb, or greater than 30 kb in length.
  • a nucleotide insertion is a single nucleotide insertion.
  • a nucleotide insertion comprises insertion of two nucleotides. [0335]
  • a nucleotide deletion is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides or 5 nucleotides in length.
  • a nucleotide deletion is 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides in length.
  • a nucleotide deletion 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 deletion is from 20-30 nucleotides, 25-40 nucleotides, 35- 50 nucleotides, 40-60 nucleotides, 50-100 nucleotides, 75-150 nucleotides, 125-250 nucleotides, 200- 300 nucleotides, 250-400 nucleotides, 300-600 nucleotides, 500-750 nucleotides, 700-1000 nucleotides, or between 1000-2000 nucleotides in length.
  • a nucleotide deletion can be between 1 and 10 kb, 1 and 20 kb, 1 and 30 kb, or greater than 30 kb in length.
  • a nucleotide deletion is a single nucleotide deletion.
  • a nucleotide deletion comprises deletion of two nucleotides.
  • Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g., mutations) in the target gene can vary.
  • 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 target gene outside of the protospacer sequence.
  • the position of a nucleotide edit incorporation in the target gene can be determined based on position of the protospacer adjacent motif (PAM).
  • the intended nucleotide edit may be installed in a sequence corresponding to the protospacer adjacent motif (PAM) sequence.
  • a nucleotide edit in the editing template is at a position corresponding to the 5’ most nucleotide of the PAM sequence.
  • a nucleotide edit in the editing template is at a position corresponding to the 3’ most nucleotide of the PAM sequence.
  • position of an intended nucleotide edit in the editing template may WSGR Docket No.59761-781.601 be referred to by aligning the editing template with the partially complementary PAM strand of the target gene, and referring to nucleotide positions on the editing strand where the intended nucleotide edit is incorporated.
  • a nucleotide edit is incorporated at a position corresponding to about 0, 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, or 40 base pairs upstream of the 5’ most nucleotide of the PAM sequence in the PAM strand of the target gene.
  • a nucleotide edit is incorporated at a position corresponding to about 0 to 2 base pairs, 0 to 4 base pairs, 0 to 6 base pairs, 0 to 8 base pairs, 0 to 10 base pairs, , 2 to 4 base pairs, 2 to 6 base pairs, 2 to 8 base pairs, 2 to 10 base pairs, 2 to 12 base pairs, 4 to 6 base pairs, 4 to 8 base pairs, 4 to 10 base pairs, 4 to 12 base pairs, 4 to 14 base pairs, 6 to 8 base pairs, 6 to 10 base pairs, 6 to 12 base pairs, 6 to 14 base pairs, 6 to16 base pairs, 8 to 10 base pairs, 8 to 12 base pairs, 8 to 14 base pairs, 8 to 16 base pairs, 8 to 18 base pairs, 10 to 12 base pairs, 10 to 14 base pairs, 10 to 16 base pairs, 10 to 18 base pairs, 10 to 20 base pairs, 12 to 14 base pairs, 12
  • the nucleotide edit is incorporated at a position corresponding to 3 base pairs upstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit in is incorporated at a position corresponding to 4 base pairs upstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit is incorporated at a position corresponding to 5 base pairs upstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit in the editing template is at a position corresponding to 6 base pairs upstream of the 5’ most nucleotide of the PAM sequence.
  • an intended nucleotide edit is incorporated at a position corresponding to about 0, 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, or 40 base pairs downstream of the 5’ most nucleotide of the PAM sequence in the PAM strand of the target gene.
  • a nucleotide edit is incorporated at a position corresponding to about 0 to 2 base pairs, 0 to 4 base pairs, 0 to 6 base pairs, 0 to 8 base pairs, 0 to 10 base pairs, , 2 to 4 base pairs, 2 to 6 base pairs, 2 to 8 base pairs, 2 to 10 base pairs, 2 to 12 base pairs, 4 to 6 base pairs, 4 to 8 base pairs, 4 to 10 base pairs, 4 to 12 base pairs, 4 to 14 base pairs, 6 to 8 base pairs, 6 to 10 base pairs, 6 to 12 base pairs, 6 to 14 base pairs, 6 to16 base pairs, 8 to 10 base pairs, 8 to 12 base pairs, 8 to 14 base pairs, 8 to 16 base pairs, 8 to 18 base pairs, 10 to 12 base pairs, 10 to 14 base pairs, 10 to 16 base pairs, 10 to 18 base pairs, 10 to 20 base pairs, 12 to WSGR Docket No.59761-781.601 14 base pairs, 12 to 16 base pairs, 12 to 18 base pairs, 12 to 20 base pairs, 12 to 22 base pairs
  • a nucleotide edit is incorporated at a position corresponding to 3 base pairs downstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 4 base pairs downstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 5 base pairs downstream of the 5’ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 6 base pairs downstream of the 5’ most nucleotide of the PAM sequence.
  • 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 of the first sequence.
  • the position of a nucleotide edit incorporation in the target gene can be determined based on position of the nick site.
  • the position of an intended nucleotide edit is 0, 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, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides away from the nick site.
  • the position of an intended nucleotide edit is 0, 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, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides upstream or downstream of the nick site on the PAM strand (or the non-target strand, or the PAM strand) of the double stranded target DNA.
  • the relative positions of the intended nucleotide edit(s) and nick site may be referred to by numbers.
  • the nucleotide immediately downstream of the nick site on a PAM strand may be referred to as at position 0.
  • the nucleotide immediately upstream of the nick site on the PAM strand (or the non-target strand, or the PAM strand) may be referred to as at position -1.
  • the nucleotides downstream of position 0 on the PAM strand can be referred to as at positions +1, +2, +3, +4, ... +n, and the nucleotides upstream of position -1 on the PAM strand may be referred to as at positions -2, -3, -4, ..., -n.
  • the number n may be referred to as the nick to edit distance.
  • the corresponding positions of the intended nucleotide edit incorporated in the target gene may also be referred to be based on the nicking position generated by a prime editor based on sequence homology and complementarity.
  • the distance between the nucleotide edits to be incorporated into the target gene and the nick generated by the prime editor may WSGR Docket No.59761-781.601 be determined when the spacer hybridizes with the search target sequence and the extension arm hybridizes with the editing target sequence.
  • the position of the nucleotide edit can be in any position downstream of the nick site on the PAM strand (or the PAM strand) generated by the prime editor, such that the distance between the nick site and the intended nucleotide edit is 0, 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, or 30 nucleotides in length.
  • the position of the nucleotide edit is 0, 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, or 30 nucleotides upstream of the nick site on the PAM strand.
  • the position of the nucleotide edit is 0, 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, or 30 nucleotides downstream of the nick site on the PAM strand.
  • the position of the nucleotide edit is 0 base pairs from the nick site on the PAM strand, that is, the editing position is at the same position as the nick site.
  • the distance between the nick site and the nucleotide edit refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the PAM strand (i.e., the “near position” of the nucleotide edit to the nick site).
  • the distance between the nick site and a PAM position edit refers to the 5’ most position of the nucleotide edit and the 5’ most position of the PAM sequence.
  • the editing template extends beyond a nucleotide edit to be incorporated to the target gene sequence.
  • the editing template comprises at least 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 or 30 base pairs 3’ to the nucleotide edit to be incorporated to the target gene sequence.
  • the editing template comprises at least 4 to 30 base pairs 3’ to the nucleotide edit to be incorporated to the target gene sequence.
  • the editing template comprises at least 4 to 25 base pairs 3’ to the nucleotide edit to be incorporated to the target gene sequence.
  • the editing template comprises at least 4 to 20 base pairs 3’ to the nucleotide edit to be incorporated to the target gene sequence. In some embodiments, the editing template comprises at least 4 to 30 base pairs 5’ to the nucleotide edit to be incorporated to the target gene sequence. In some embodiments, the editing template comprises at least 4 to 25 base pairs 5’ to the nucleotide edit to be incorporated to the target gene sequence. In some embodiments, the editing template comprises at least 4 to 20 base pairs 5’ to the nucleotide edit to be incorporated to the target gene 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 WSGR Docket No.59761-781.601 comprises an 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”).
  • the editing template of a PEgRNA may encode a new single stranded DNA (e.g. by reverse transcription) to replace a target sequence in the target gene.
  • the editing target sequence in the PAM strand of the target gene is replaced by the newly synthesized strand, and the nucleotide edit(s) are incorporated in the region of the target gene.
  • the target gene is a target gene.
  • the editing template of the PEgRNA encodes a newly synthesized single stranded DNA that comprises a wild type target gene sequence.
  • the newly synthesized DNA strand replaces the editing target sequence in the target gene, wherein the editing target sequence (or the endogenous sequence complementary to the editing target sequence on the target strand of the target gene) comprises a mutation compared to a wild type target gene.
  • the editing target sequence comprises a mutation in an intron of the target gene as compared to a wild type target gene.
  • the editing target sequence comprises a mutation in an intron of the target gene that results in altered or aberrant splicing of a transcript encoded by the target gene compared to a transcript encoded by a wild type target gene.
  • the editing template comprises one or more intended nucleotide edits compared to the sequence on the target strand of the target gene that is complementary to the editing target sequence.
  • the editing template encodes a single stranded DNA that comprises one or more intended nucleotide edits compared to the editing target sequence.
  • the single stranded DNA replaces the editing target sequence by prime editing, thereby incorporating the one or more intended nucleotide edits.
  • incorporation of the one or more intended nucleotide edits corrects the mutation in the editing target sequence to wild type nucleotides at corresponding positions in the target gene.
  • “correcting” a mutation means restoring a wild type sequence at the place of the mutation in the double stranded target DNA, e.g., target gene, by prime editing.
  • the editing template comprises and/or encodes a wild type target gene sequence.
  • incorporation of the one or more intended nucleotide edits does not correct the mutation in the editing target sequence to wild type sequence but allows for expression of a functional protein encoded by the target gene.
  • the editing template comprises one or more intended nucleotide edits compared to the sequence on the target strand of the target gene that is complementary to the editing WSGR Docket No.59761-781.601 target sequence, wherein the one or more intended nucleotide edits is a single nucleotide substitution, polynucleotide substitution, nucleotide insertion, or nucleotide deletion.
  • the intended nucleotide edit in the editing template comprises a single nucleotide substitution, polynucleotide substitution, nucleotide insertion, or nucleotide deletion compared to the sequence on the target strand of the target gene that is complementary to the editing target at a position corresponding to a mutation in target gene, wherein the editing target sequence is on the sense strand of the target gene.
  • a guide RNA core also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence
  • a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas12i2) 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 DNA nickase of the prime editor.
  • the PEgRNA comprises a guide RNA (gRNA) core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor.
  • gRNA guide RNA
  • the gRNA core comprises a first gRNA core sequence comprising a 5’ half of the gRNA core and a second gRNA core sequence comprising a 3’ half of the gRNA core
  • the PEgRNA comprises, in 5’ to 3’ order: the spacer, the first gRNA core sequence, the editing template, the PBS, the tag sequence, and the second gRNA core sequence.
  • the 5’half and the 3’half can form a functional gRNA core for association/binding with a programmable DNA binding protein, e.g., a Cas12i2 protein.
  • a programmable DNA binding protein e.g., a Cas12i2 protein.
  • 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 can be ises 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 MS2cp protein).
  • 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.
  • the WSGR Docket No.59761-781.601 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. In some embodiments, the PEgRNA RNA structures, 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 or a nick guide RNA can be chemically synthesized, or can be assembled or cloned and transcribed from a DNA sequence, e.g., a plasmid DNA sequence, or by any RNA oligonucleotide synthesis method known in the art.
  • DNA sequence that encodes a PEgRNA (or ngRNA) can be designed to append one or ick guide RNA) encoding sequence to enhance PEgRNA transcription. For example, in some embodiments, a DNA sequence that encodes end.
  • the PEgRNA can comprise an (or nick guide RNA) can be designed to append a sequence that enhances transcription, e.g., a Kozak sequence
  • the PEgRNA (or nick guide RNA) can comprise an appended sequence CACC or end.
  • the PEgRNA (or nick guide RNA) can comprise an sequence AACAUUGACGCGUCUCUACGUGGGGGCGCG.
  • a prime editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA).
  • the non-PAM strand of a double stranded target DNA in the target gene may be nicked by a CRISPR-Cas nickase directed by an ngRNA.
  • the nick on the non-PAM strand directs endogenous DNA repair machinery to use the PAM strand as a template for repair of the non- PAM strand, which may increase efficiency of prime editing.
  • the non-PAM strand is nicked by a prime editor localized to the non-PAM strand by the ngRNA.
  • PEgRNA systems comprising at least one PEgRNA and at least one ngRNA.
  • the ngRNA is a guide RNA which contains a variable spacer sequence and a guide RNA scaffold or core region that interacts with the DNA binding domain, e.g., Cas12i2 of the prime editor.
  • the ngRNA comprises a spacer sequence (referred to herein as an ng spacer, or a second spacer) that is substantially complementary to a second search target sequence (or ng search target sequence), which is located on the PAM strand, or the non-target strand.
  • the ng search target sequence recognized by the ng spacer and the search target sequence recognized by the spacer sequence of the PEgRNA are on opposite strands of the double stranded target DNA of target gene.
  • the ng search target sequence is located on the non-target strand, within 10 base pairs to 100 base pairs of an intended nucleotide edit incorporated by the PEgRNA on the PAM strand.
  • the ng target search target sequence is within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp of an intended nucleotide edit incorporated by the PEgRNA on the PAM strand.
  • the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart from each other.
  • the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp apart from each other.
  • an ng spacer sequence is complementary to, and may hybridize with the second search target sequence only after an intended nucleotide edit has been incorporated on the PAM strand, by the editing template of a PEgRNA.
  • the ngRNA comprises a spacer sequence that matches only the PAM strand after incorporation of the nucleotide edits, but not the endogenous target gene sequence on the PAM strand.
  • an intended nucleotide edit is incorporated within the ng search target sequence.
  • the intended nucleotide edit is incorporated within about 1-10 nucleotides of the position corresponding to the PAM of the ng search target sequence.
  • a PEgRNA comprises an additional secondary structure at the 5’ end. In some embodiments, a PEgRNA (or ngRNA) comprises an additional secondary structure at the 3’ end. [0359] In some embodiments, the secondary structure comprises a pseudoknot. In some embodiments, 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).
  • M-MLV Moloney murine leukemia virus
  • the secondary structure comprises a nucleotide sequence selected from the group consisting of GGGUCAGGAGCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGCAACC (SEQ ID NO: 935), GUCAGGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGCAAC WSGR Docket No.59761-781.601 CC (SEQ ID NO: 859), GGGUCAGGAGCCCCCCCUGAACCCAGGAAAACCCUCAAAGUCGGGGGGCAACCC (SEQ ID NO: 860), GGGUCAGGAGCCCCCCCCCUGCACCCAGGAAAACCCUCAAAGUCGGGGGGCAACCC (SEQ ID NO: 861), GGGUCAGGAGCCCCCCCCCUGCACCCAGGAUAACCCUCAAAGUCGGGGGGCAACCC (SEQ ID NO: 862), GUCAGGGUCAGGAGCCCCCCCCCUGAACCCAGGAAAACCCUCAAAG
  • the secondary structure comprises a nucleotide sequence of GGGUCAGGAGCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGC (SEQ ID NO: 865), 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. In some embodiments, the secondary structure comprises a G-quadruplex.
  • the secondary structure comprises a nucleotide sequence selected from the group consisting of gq2 (UGGUGGUGGUGGU) (SEQ ID NO: 866), stk40 (GGGACAGGGCAGGGACAGGG) (SEQ ID NO: 867), apc2 (GGGUCCGGGUCUGGGUCUGGG) (SEQ ID NO: 868), stard3 (GGGCAGGGUCUGGGCUGGG) (SEQ ID NO: 869), tns1 (GGGCUGGGAUGGGAAAGGG) (SEQ ID NO: 870), ceacam4 (GGGCUCUGGGUGGGCCGGG) (SEQ ID NO: 871), erc1 (GGGCUGGGCUGGGCAGGG) (SEQ ID NO: 872), pitpnm3 (GGGUGGGCUGGGAAGGG) (SEQ ID NO: 873), rlf (GGGAGGGAGGGCUAGGG) (SEQ ID NO: 874), ube3c (GG
  • the secondary structure comprises aP4-P6 domain of a Group I intron.
  • the secondary structure comprises the nucleotide sequence of GGAAUUGCGGGAAAGGGGUCAACAGCCGUUCAGUACCAAGUCUCAGGGGAAACUUUG AGAUGGCCUUGCAAAGGGUAUGGUAAUAAGCUGACGGACAUGGUCCUAACCACGCAG CCAAGUCCUAAGUCAACAGAUCUUCUGUUGAUAUGGAUGCAGUUCA (SEQ ID NO: 878), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
  • the secondary structure comprises a riboswitch aptamer. In some embodiments, the secondary structure comprises a riboswitch aptamer derived from a prequeosine-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a modified prequeosine-1 riboswitch aptamer.
  • the secondary structure comprises a nucleotide sequence selected from the group consisting of UUGACGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAAA (SEQ ID NO: 879), UUGACGCGGUUCUAUCUACUUACGCGUUAAACCAACUAGAAA (SEQ ID NO: 880), CGCGAGUCUAGGGGAUAACGCGUUAAACUUCCUAGAAGGCGGUU (SEQ ID NO: 881), CGCGGAUCUAGAUUGUAACGCGUUAAACCAUCUAGAAGGCGGUU (SEQ ID NO: 882), CGCGUCGCUACCGCCCGGCGCGUUAAACACACUAGAAGGCGGUU (SEQ ID NO: 883), and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID NO: 885), 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 selected from the group consisting of UUGACGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAAA (SEQ ID NO: 879), CGCGAGUCUAGGGGAUAACGCGUUAAACUUCCUAGAAGGCGGUU (SEQ ID NO: 881), CGCGGAUCUAGAUUGUAACGCGUUAAACCAUCUAGAAGGCGGUU (SEQ ID NO: 882), CGCGUCGCUACCGCCCGGCGCGUUAAACACACUAGAAGGCGGUU (SEQ ID NO: 883), and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID NO: 885), 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 and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID NO: 885), 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. In some embodiments, the linker is 10 to 20 nucleotides in length.
  • the linker is 15 to 25 nucleotides in length. In some embodiments, the linker is 8 nucleotides in length. [0364] In some embodiments, 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 WSGR Docket No.59761-781.601 designed to minimize base pairing between the linker and the sequence of the RNA secondary structure.
  • 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, as described in Lorenz, R. et al. Vienna RNA package 2.0. Algorithms Mol. Biol.6, incorporated by reference in its entirety herein, under standard parameters (37 °C, 1 M NaCl, 0.05 M M MgCl2).
  • the PEgRNA comprises an RNA secondary structure and/or a linker disclosed in Nelson et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol.
  • a PEgRNA is transcribed from a nucleotide encoding the PEgRNA, for example, a DNA plasmid encoding the PEgRNA.
  • the PEgRNA comprises 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.
  • the PEgRNA comprises an element for nuclear expression (ENE) from MALAT1 lnc RNA.
  • the PEgRNA comprises an ENE element from Kaposi’s sarcoma-associated herpesvirus (KSHV).
  • the PEgRNA comprises a 3’ box of a U1 snRNA. In some embodiments, the PEgRNA forms a circular RNA. [0367] 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. In some embodiments, the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, e.g., initiation of retroviral transcription. In some embodiments, 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.
  • PPT polypurine tract
  • 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.
  • a PEgRNA comprises the sequence GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGC GAAUGGGAC (SEQ ID NO: 886) at the 3’ end.
  • a PEgRNA comprises the structure [spacer]-[gRNA core]-[editing template]-[PBS]- GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGC GAAUGGGAC (SEQ ID NO: 886), or [spacer]-[gRNA core]-[editing template]-[PBS]- GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGC GAAUGGGAC-(U)n (SEQ ID NO: 900), wherein n is an integer between 3 and 7.
  • the structure derived from hepatitis D virus (HDV) is italicized.
  • the PEgRNA comprises the sequence GGUGGGAGACGUCCCACC (SEQ ID NO: 880) at the 5’ end and/or the sequence UGGGAGACGUCCCACC (SEQ ID NO: 901) at the 3’ end.
  • the PEgRNA comprises the following structure (M-MLV kissing loop): GGUGGGAGACGUCCCACC (SEQ ID NO: 880)-[spacer]-[gRNA core]-[editing template]- [PBS]-UGGGAGACGUCCCACC (SEQ ID NO: 901), or GGUGGGAGACGUCCCACC (SEQ ID NO: 880)-[spacer]-[gRNA core]-[editing template]-[PBS]-UGGGAGACGUCCCACC-(U)n (SEQ ID NO: 902), wherein n is an integer between 3 and 7.
  • the kissing loop structure is italicized.
  • the PEgRNA comprises the sequence GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 888) at the 5’ end and/or the sequence CCAUCAGUUGACACCCUGAGG (SEQ ID NO: 889) at the 3’ end.
  • the PEgRNA comprises the following structure (VS ribozyme kissing loop): GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 888)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG (SEQ ID NO: 889), or GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 888)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG-(U)n (SEQ ID NO: 903), wherein n is an integer between 3 and 7.
  • the PEgRNA comprises the sequence GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 890) at the 5’ end and/or the sequence CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID NO: 891) at the 3’ end.
  • the PEgRNA comprises the following structure (tetraloop and receptor): GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 890)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID NO: 891), or GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 890)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG-(U)n (SEQ ID NO: 904), wherein n is an integer between 3 and 7.
  • the PEgRNA comprises the sequence GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGG CGAAUGGGAC (SEQ ID NO: 886) or WSGR Docket No.59761-781.601 UCUGCCAUCAAAGCUGCGACCGUGCUCAGUCUGGUGGGAGACGUCCCACCGGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGG AC (SEQ ID NO: 892) at the 3’ end.
  • a PEgRNA comprises a gRNA core comprises a sequence selected from Table 6.
  • a PEgRNA and/or an ngRNA 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 and/or ngRNAs 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 the disclosure may further comprise nucleotides added to the 5’ of the PEgRNAs.
  • the PEgRNA further comprises 1, 2, or 3 additional nucleotides added to the 5’ end.
  • the additional nucleotides can be guanine, cytosine, adenine, or uracil.
  • the additional nucleotide at the 5’ end of the PEgRNA is a guanine or cytosine. In some embodiments, the additional nucleotides can be chemically or biologically modified. [0376] In some embodiments, the PEgRNAs provided in the disclosure may further comprise nucleotides to the 3’ of the PEgRNAs. In some embodiments, the PEgRNA further comprises 1, 2, or 3 additional nucleotides to the 3’ end. The additional nucleotides can be guanine, cytosine, adenine, or uracil. In some embodiments, the additional nucleotides at the 3’ end of the PEgRNA is a polynucleotide comprising at least 1 uracil.
  • the additional nucleotides can be chemically or biologically modified.
  • a PEgRNA or ngRNA is produced by transcription from a template nucleotide, for example, a template plasmid.
  • a polynucleotide encoding the PEgRNA or ngRNA is appended with one or more additional nucleotides that improves PEgRNA or ngRNA function or expression, e.g., expression from a plasmid that encodes the PEgRNA or ngRNA.
  • a polynucleotide encoding a PEgRNA or ngRNA is appended with one or more additional nucleotides at the 5’ end or at the 3’ end.
  • the polynucleotide encoding the PEgRNA or ngRNA 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 or ngRNA is appended with nucleotide sequence CACC at the 5’ end.
  • the polynucleotide encoding the PEgRNA or ngRNA 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 WSGR Docket No.59761-781.601 Thymine.
  • the polynucleotide encoding the PEgRNA or ngRNA is appended with additional nucleotide sequence TTTT, TTTTTTT, TTTTT, or TTTT at the 3’ end.
  • the PEgRNA or ngRNA comprises the appended nucleotides from the transcription template.
  • the PEgRNA or ngRNA further comprises nucleotide sequence UUUUUU, UUUUU, UUUUU, or UUUU at the 3’ end.
  • the PEgRNAs and/or ngRNAs provided in this disclosure may have undergone a chemical or biological modification. Modifications may be made at any position within a PEgRNA or ngRNA and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA or ngRNA.
  • chemical modifications can be structure guided modifications.
  • a chemical modification is at the 5’ end and/or the 3’ end of a PEgRNA.
  • a chemical modification is at the 5’ end and/or the 3’ end of a ngRNA.
  • a chemical modification can be within the spacer sequence, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA.
  • a chemical modification may be within the spacer sequence or the gRNA core of a PEgRNA or a ngRNA.
  • a chemical modification can be within the 3’ most nucleotides of a PEgRNA or ngRNA.
  • a chemical modification can be within the 3’ most end of a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modification can be within the 5’ most end of a PEgRNA or ngRNA.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 or more chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5’ end.
  • a PEgRNA or WSGR Docket No.59761-781.601 ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5’ end.
  • a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 5’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA 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 or ngRNA 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 chemical modification can also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA and/or ngRNA (e.g., modifications to one or both of the 3' and 5' ends of a guide RNA molecule).
  • Prime editing compositions 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).
  • agent e.g., a protein or a complementary nucleic acid molecule
  • Prime editing compositions [0380] Disclosed herein, in some embodiments, are compositions, systems, and methods using a prime editing composition.
  • the term “prime editing composition” or “prime editing system” refers to compositions involved in the method of prime editing as described herein.
  • a prime editing WSGR Docket No.59761-781.601 composition may include a prime editor provided herein and a PEgRNA.
  • a prime editing composition may further comprise additional elements, such as second strand nicking ngRNAs.
  • a prime editing composition comprises a prime editor fusion protein complexed with a PEgRNA and optionally complexed with a ngRNA.
  • the prime editing composition comprises a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through a 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 a PEgRNA.
  • a prime editing composition comprises a PEgRNA and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein.
  • a prime editing composition comprises a PEgRNA, a ngRNA, 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 PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas12i2 nickase, of the prime editor. In some embodiments, the PEgRNA of a prime editing composition complexes with the DNA binding domain of a prime editor and directs the prime editor to the target DNA.
  • a prime editing composition comprises one or more polynucleotides that encode prime editor components and/or PEgRNA or ngRNAs. In some embodiments, 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, and (ii) a PEgRNA or a polynucleotide encoding the PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iii) an ngRNA or a polynucleotide encoding the ngRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas12i2 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, and (iii) a PEgRNA or a polynucleotide encoding the PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas12i2 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iv) an ngRNA or a polynucleotide encoding the ngRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas12i2 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEg
  • a prime editing composition comprises a first prime editing guide RNA (PEgRNA), a second PEgRNA, and a prime editor.
  • 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.
  • 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 Cas12i2 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 consists of (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 DNA binding domain of a prime editor, e.g., a Cas12i2 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 Cas12i2 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii
  • a prime editing composition consists of (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas12i2 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 consists of (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas12i2 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase,
  • the polynucleotide encoding the DNA biding domain or the polynucleotide encoding the DNA polymerase domain further encodes an additional polypeptide domain, e.g., a recombinase domain, or an RNA-protein recruitment domain, such as a MS2 coat protein domain.
  • 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 PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) an ngRNA or a polynucleotide encoding the ngRNA.
  • 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 Cas12i2 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 PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) a ngRNA or a polynucleotide encoding the ngRNA.
  • 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 WSGR Docket No.59761-781.601 temporally regulated by controlling the timing in which the vectors are delivered.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA may be delivered simultaneously.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA 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 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. In some embodiments, the element may be within the 3 ⁇ UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription. [0391] In some embodiments, the element may include at least one AU-rich element (ARE).
  • 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 (ARE-BPs) 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).
  • 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 WSGR Docket No.59761-781.601 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 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.
  • Pharmaceutical compositions [0394] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, prime editing complexes, and/or the fusion protein-guide polynucleotide complexes described herein.
  • 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).
  • pharmaceutically-acceptable carrier means a pharmaceutically acceptable material, composition or 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).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • 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, physiologic pH, etc.).
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanthin; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
  • compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0.
  • the pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine.
  • the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions.
  • a predetermined level such as in the range of about 5.0 to about 8.0
  • pH buffering compounds include, but are not limited to, imidazole and acetate ions.
  • the pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.
  • compositions can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals.
  • the osmotic modulating agent can be an agent that does not chelate calcium ions.
  • the osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation.
  • osmotic modulating agents include, but are not limited to, salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents.
  • the osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.
  • the pharmaceutical composition comprising any of the prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, prime editing complexes, and/or the fusion protein-guide polynucleotide complexes described herein may be used in vitro or in vivo.
  • the pharmaceutical composition or any components thereof are delivered to cells (e.g., mammalian cells, e.g., human cells), e.g., in vitro or in vivo.
  • the pharmaceutical composition is formulated for delivery to a subject, e.g., for prime editing.
  • Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, WSGR Docket No.59761-781.601 intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
  • the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site).
  • the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • the pharmaceutical composition described herein is delivered in a controlled release system.
  • a pump can be used.
  • polymeric materials can be used.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human.
  • pharmaceutical composition for administration by injection are solutions in sterile isotonic use as solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • a pharmaceutical composition for systemic administration can be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution.
  • the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.
  • the pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration.
  • the particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein.
  • Compounds can be entrapped in“stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid and stabilized by a polyethyleneglycol (PEG) coating.
  • SPLP stabilized plasmid-lipid particles
  • DOPE fusogenic lipid dioleoylphosphatidylethanolamine
  • PEG polyethyleneglycol
  • Positively charged lipids such as N-[l-(2,3- dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles.
  • the pharmaceutical composition described herein can be administered or packaged as a unit dose, for example, in reference to a pharmaceutical composition to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
  • the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a WSGR Docket No.59761-781.601 second container containing a pharmaceutically acceptable diluent (e.g., sterile used for reconstitution or dilution of the lyophilized compound of the invention.
  • a pharmaceutically acceptable diluent e.g., sterile used for reconstitution or dilution of the lyophilized compound of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Any of the prime editors, fusion proteins, PEgRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition.
  • the pharmaceutical composition comprises any of the prime editors or fusion proteins provided herein.
  • the pharmaceutical composition comprises any of the complexes provided herein.
  • the pharmaceutical composition comprises a ribonucleoprotein complex comprising a prime editor that forms a complex with a PEgRNA and a cationic lipid.
  • compositions comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient.
  • Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
  • 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 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.
  • the compositions, as described above can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition
  • the prime editing method comprises contacting a target gene, with a PEgRNA and a prime editor (PE) polypeptide described herein.
  • the WSGR Docket No.59761-781.601 target gene is double stranded, and comprises two strands of DNA complementary to each other.
  • the contacting with a PEgRNA and the contacting with a prime editor are performed sequentially.
  • the contacting with a prime editor is performed after the contacting with a PEgRNA.
  • the contacting with a PEgRNA is performed after the contacting with a prime editor. In some embodiments, the contacting with a PEgRNA, and the contacting with a prime editor are performed simultaneously. In some embodiments, the PEgRNA and the prime editor are associated in a complex prior to contacting a target gene. [0406] In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a target strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the target strand of the target gene upon contacting with the PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of a spacer sequence of the PEgRNA to a search target sequence with the search target sequence on the target strand of the target gene upon said contacting of the PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, upon the contacting of the PE composition with the target gene.
  • the DNA binding domain of the PE associates with the PEgRNA.
  • the PE binds the target gene, directed by the PEgRNA. Accordingly, in some embodiments, the contacting of the target gene result in binding of a DNA binding domain of a prime editor of the target gene directed by the PEgRNA.
  • contacting the target gene with the prime editing composition results in a nick in a PAM strand of the target gene by the prime editor upon contacting with the target gene, thereby generating a nick on the PAM strand of the target gene.
  • contacting the target gene with the prime editing composition results in a single-stranded DNA comprising a free 3 ⁇ end at the nick site of the PAM strand of the target gene.
  • contacting the target gene with the prime editing composition results in a nick in the PAM strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3 ⁇ end at the nick site.
  • contacting the target gene with the prime editing composition results in a nick in a target strand of the target gene by the prime editor upon contacting with the target gene, thereby generating a nick on the target strand of the target gene.
  • contacting the target gene with the prime editing composition results in a single- stranded DNA comprising a free 3 ⁇ end at the nick site of the target strand of the target gene.
  • contacting the target gene with the prime editing composition results in a nick in the target strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3 ⁇ end at the nick site.
  • 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 Cas12i2. In some embodiments, the DNA binding domain of the prime editor WSGR Docket No.59761-781.601 is a Cas12i2 nickase. In some embodiments, the DNA binding domain of the prime editor is a nuclease active Cas12i2. [0409] In some embodiments, contacting the target gene with the prime editing composition results in hybridization of the PEgRNA with the 3’ end 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).
  • contacting the target gene with the prime editing composition generates an edited single stranded DNA that is coded by the editing template of the PEgRNA by DNA polymerase mediated polymerization from the 3’ free end of the single-stranded DNA at the nick site.
  • the editing template of the PEgRNA comprises one or more intended nucleotide edits compared to endogenous sequence of the target gene.
  • the intended nucleotide edits are incorporated in the target gene, by excision of the 5’ single stranded DNA of the nicked strand (either the target strand or the PAM strand) of the target gene generated at the nick site and DNA repair.
  • the intended nucleotide edits are incorporated in the target gene by excision of the editing target sequence and DNA repair.
  • excision of the 5’ single stranded DNA of the nicked strand (either the target strand or the PAM strand) PAM strand generated at the nick site is by a flap endonuclease.
  • the flap nuclease is FEN1.
  • the method further comprises contacting the target gene with a flap endonuclease.
  • the flap endonuclease is provided as a part of a prime editor fusion protein.
  • the flap endonuclease is provided in trans.
  • contacting the target gene with the prime editing composition generates a mismatched heteroduplex comprising the edited strand of the target gene that comprises the edited single stranded DNA, and the unedited target strand of the target gene.
  • the endogenous DNA repair and replication may resolve the mismatched edited DNA to incorporate the nucleotide change(s) to form the desired edited target gene.
  • the method further comprises contacting the target gene, with a nick guide (ngRNA) disclosed herein.
  • the ngRNA comprises a spacer that binds a second search target sequence on the unedited strand of the target gene.
  • the contacted ngRNA directs the PE to introduce a nick in the target strand of the target gene.
  • the nick on the unedited strand results in endogenous DNA repair machinery to use edited strand to repair the unedited strand, thereby incorporating the intended nucleotide edit in both strand of the target gene and modifying the target gene.
  • the ngRNA comprises WSGR Docket No.59761-781.601 a spacer sequence that is complementary to, and may hybridize with, the second search target sequence on the unedited strand only after the intended nucleotide edit(s) are incorporated in the unedited strand of the target gene.
  • the target gene is contacted by the ngRNA, the PEgRNA, and the PE simultaneously. In some embodiments, the ngRNA, the PEgRNA, and the PE form a complex when they contact the target gene. In some embodiments, the target gene is contacted with the ngRNA, the PEgRNA, and the prime editor sequentially. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA after contacting the target gene with the PE. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA before contacting the target gene with the prime editor. [0414] 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. In some embodiments, the prime editor is different for each of the PEgRNA-prime editor complexes. In some embodiments, 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. In some embodiments, 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. In some embodiments, each of the two PEgRNAs comprises a spacer complementary to a separate search target sequence.
  • 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.
  • a first PEgRNA comprises a first editing template.
  • 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.
  • 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.
  • 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
  • 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 region of complementarity between the first editing template and the second editing template comprises a nucleotide sequence that is exogenous to the double stranded target DNA or target gene.
  • 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. In certain embodiments, 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. [0418] In certain embodiments, 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), and 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 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 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 nucleotides compared to the endogenous sequence of the double stranded target gene.
  • the target gene is in a cell. Accordingly, also provided herein are methods of modifying a cell.
  • the prime editing method comprises introducing a PEgRNA, a prime editor, and/or a ngRNA into the cell that has the target gene.
  • the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA.
  • the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex prior to the introduction into the cell.
  • the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex after the introduction into the cell.
  • the prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell by any delivery approaches described herein WSGR Docket No.59761-781.601 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.
  • the prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell simultaneously or sequentially.
  • the prime editing method comprises introducing into the cell a PEgRNA or a polynucleotide encoding the PEgRNA, a prime editor polynucleotide encoding a prime editor polypeptide, and optionally an ngRNA or a polynucleotide encoding the ngRNA.
  • the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell simultaneously.
  • the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA 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 PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA.
  • the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA are introduced into the cell.
  • the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA 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, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA integrate into the genome of the cell after being introduced into the cell.
  • the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA are introduced into the cell for transient expression. Accordingly, also provided herein are cells modified by prime editing. [0423]
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a non-human primate cell, bovine cell, porcine cell, rodent or mouse cell.
  • the cell is a human cell.
  • the cell is a primary cell. In some embodiments, the cell is a human primary cell. In some embodiments, the cell is a progenitor cell. In some embodiments, the cell is a WSGR Docket No.59761-781.601 human progenitor cell. In some embodiments, the cell is a human cell from an organ. In some embodiments, the cell is a primary human cell derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a megakaryocyte erythroid progenitor cell.
  • iPSC induced human pluripotent stem cell
  • the cell is a muscle cell (e.g., cardiac muscle cells, smooth muscle cells, hepatocytes), hematopoietic stem cell (HSC), hematopoietic stem progenitor cell (HSPC), a fibroblast, a keratinocyte, an epithelial cell (e.g., mammary epithelial cells, intestinal epithelial cells), an endothelial cell, a glial cell, a neural cell, a cell of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.
  • the human cell is a human stem cell.
  • the human cell is a human progenitor cell.
  • the human cell is a pluripotent human cell (e.g., a pluripotent stem cell).
  • the cell is a human HSPC.
  • the cell is a progenitor cell.
  • the cell is a stem cell.
  • the cell is an induced pluripotent stem cell.
  • the cell is an embryonic stem cell.
  • the cell is a hematopoietic progenitor cell.
  • the cell is a hematopoietic stem cell.
  • the cell is a hematopoietic stem or progenitor cell (HSPC).
  • the cell is a fibroblast. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a hematopoietic stem cell (HSC). In some embodiments, the cell is a hematopoietic progenitor cell (HPC). In some embodiments, the cell is a human HSPC. In some embodiments, the cell is a human HPC. In some embodiments, the cell is a human HSC. In some embodiments, the cell is a long term (LT)-HSC. In some embodiments, the cell is a short-term (ST)-HSC. In some embodiments, the cell is a myeloid progenitor cell.
  • HSC hematopoietic stem cell
  • HPC hematopoietic progenitor cell
  • HPC hematopoietic progenitor cell
  • the cell is a human HSPC.
  • the cell is a human HPC.
  • the cell is a human HSC.
  • the cell is a lymphoid progenitor cell. In some embodiments, the cell is a granulocyte monocyte progenitor cell. [0426] In some embodiments, the cell is a human progenitor cell. In some embodiments, the cell is a human stem cell. in some embodiments, the cell is an induced human pluripotent stem cell. In some embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human fibroblast. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a human primary cell. In some embodiments, the cell is a differentiated cell.
  • the cell edited by prime editing can be differentiated into or give rise to recovery of a population of cells, e.g., neutrophils, platelets, red blood cells, monocytes, macrophages, antigen-presenting cells, microglia, osteoclasts, dendritic cells, inner ear cell, inner ear support cell, cochlear cell and/or lymphocytes.
  • the target gene edited by prime editing is in a chromosome of the cell.
  • the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells.
  • the intended nucleotide edits introduced to the cell by the prime editing compositions and methods are such that the cell and progeny of the cell also include the intended nucleotide edits.
  • the cell is autologous, allogeneic, or xenogeneic to a subject.
  • the cell is from or derived from a subject.
  • the cell is from or derived from a human subject.
  • 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, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA into a plurality or a population of cells that comprise the target gene.
  • the population of cells is of the same cell type.
  • the population of cells is of the same tissue or organ.
  • the population of cells is heterogeneous.
  • the population of cells is homogeneous.
  • the population of cells is from a single tissue or organ, and the cells are heterogeneous.
  • the introduction into the population of cells is ex vivo.
  • the introduction into the population of cells is in vivo, e.g., into a human subject.
  • the target gene is in a genome of each cell of the population.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA 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, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in a plurality of the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in sufficient number of cells such that the disease or disorder is treated, prevented or ameliorated.
  • 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 target gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a target 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 WSGR Docket No.59761-781.601 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 methods 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. [0431] In some embodiments, 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 target cell, e.g., a primary cell, relative to a suitable control.
  • a target cell e.g., a primary cell
  • 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 hepatocyte relative to a corresponding control hepatocyte.
  • the hepatocyte is a human hepatocyte.
  • the prime editing compositions provided herein are capable of incorporated one or more intended nucleotide edits without generating a significant proportion of indels.
  • Indel(s) 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. In some embodiments, 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 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 to a prime editing composition.
  • the prime editing compositions provided herein are capable of incorporated 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 cell.
  • 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 cell.
  • 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 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 1% in a target cell, e.g., a human 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 cell.
  • 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 cell. [0435] 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 target cell, e.g., a human 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 cell.
  • 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 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 cell.
  • 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 cell.
  • 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 cell. [0437] 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 target cell, e.g., a human 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 cell.
  • 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 cell. [0438] 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 target cell, e.g., a human 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 cell.
  • 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 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 cell.
  • 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 cell.
  • 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 cell. [0440] 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 1% in a target cell, e.g., a human cell. 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 target cell, e.g., a human 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 cell. [0441] 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 target cell, e.g., a human 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 cell.
  • 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 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 cell.
  • 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 cell.
  • 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 cell. [0443] 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 target cell, e.g., a human 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 cell.
  • 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 cell. [0444] 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 1% in a target cell, e.g., a human 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 cell.
  • 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 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 cell.
  • 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 cell.
  • 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 cell. [0446] 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% in a target cell, e.g., a human cell. 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% in a target cell, e.g., a human 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 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 gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a 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 gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a gene within the genome of a cell
  • the prime editing composition described herein result 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 compositions e.g., PEgRNAs and prime editors as described herein
  • prime editing methods disclosed herein can be used to edit a target gene.
  • the target gene comprises a mutation compared to a wild type gene.
  • the mutation is associated a disease.
  • the target gene comprises WSGR Docket No.59761-781.601 an editing target sequence that contains the mutation associated with a disease.
  • the mutation is in a coding region of the target gene.
  • the mutation is in an exon of the target gene.
  • the prime editing method comprises contacting a target gene with a prime editing composition comprising a prime editor, a PEgRNA, and/or a ngRNA. In some embodiments, contacting the target gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene.
  • the incorporation is in a region of the target gene that corresponds to an editing target sequence in the gene.
  • the one or more intended nucleotide edits comprises a single nucleotide substitution, an insertion, a deletion, or any combination thereof, compared to the endogenous sequence of the target gene.
  • incorporation of the one or more intended nucleotide edits results in replacement of one or more mutations with the corresponding sequence that encodes a wild type protein.
  • incorporation of the one or more intended nucleotide edits results in replacement of the one or more mutations with the corresponding sequence in a wild type gene.
  • incorporation of the one more intended nucleotide edits results in correction of a mutation in the target gene.
  • the target gene comprises an editing template sequence that contains the mutation.
  • contacting the target gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene, which corrects the mutation in the editing target sequence (or a double stranded region comprising the editing target sequence and the complementary sequence to the editing target sequence on a target strand) in the target gene.
  • incorporation of the one more intended nucleotide edits results in correction of a mutation in.
  • incorporation of the one more intended nucleotide edits results in correction of a gene sequence and restores wild type expression and function of the protein.
  • the target gene is in a target cell.
  • the methods of the present disclosure comprise introducing a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA into the target cell that has the target gene to edit the target gene, thereby generating an edited cell.
  • the target cell is a mammalian cell.
  • the target cell is a human cell. In some embodiments, the target cell is a primary cell. In some embodiments, the target cell is a human primary cell. In some embodiments, the target cell is a progenitor cell. In some embodiments, the target cell is a human progenitor cell. In some embodiments, the target cell is a stem cell. In some embodiments, the cell is a human cell from an organ. In some embodiments, the cell is a primary human cell derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a megakaryocyte erythroid progenitor cell.
  • iPSC induced human pluripotent stem cell
  • the cell is a muscle cell WSGR Docket No.59761-781.601 (e.g., cardiac muscle cells, smooth muscle cells) a hepatocyte, hematopoietic stem cell (HSC), hematopoietic stem progenitor cell (HSPC), a fibroblast, a keratinocyte, an epithelial cell (e.g., mammary epithelial cells, intestinal epithelial cells), an endothelial cell, a glial cell, a neural cell, a cell of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.
  • the human cell is a human stem cell.
  • the human cell is a human progenitor cell. In some embodiments, the human cell is a pluripotent human cell (e.g., a pluripotent stem cell).
  • the target cell is a human stem cell. In some embodiments, the target cell is a hepatocyte. In some embodiments, the target cell is a human hepatocyte. In some embodiments, the target cell is a primary human hepatocyte derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia.
  • the cell is a neuron from basal ganglia of a subject. In some embodiments, the cell is a neuron in the basal ganglia of a subject.
  • components of a prime editing composition described herein are provided to a target cell in vitro. In some embodiments, 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. [0453] In some embodiments, incorporation of the one or more intended nucleotide edits in the target gene that comprises one or more mutations restores wild type expression and function of protein encoded by the gene.
  • the target gene encodes at least one mutation as compared to the wild type protein prior to incorporation of the one or more intended nucleotide edits.
  • expression and/or function of protein may be measured when expressed in a target cell.
  • incorporation of the one or more intended nucleotide edits in the target gene comprising one or more mutations lead to a fold change in a level of gene expression, protein expression, or a combination thereof.
  • a change in the level of gene expression 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 greater as compared to expression in a suitable control cell not introduced with a prime editing composition described herein.
  • incorporation of the one or more intended nucleotide edits in the target gene that comprises one or more mutations restores wild type expression of protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, o99% or more as compared to wild type expression of the protein in a suitable control cell that comprises a wild type gene.
  • an expression increase can be measured by a functional assay.
  • protein expression can be measured using a protein assay.
  • protein expression can be measured using antibody testing.
  • protein expression can be measured using ELISA, mass spectrometry, Western blot, sodium dodecyl sulfate WSGR Docket No.59761-781.601 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.
  • a disease or disorder that involve the introduction of a prime editor into a disease-associated or disease-causing gene, or into a regulatory sequence (e.g., a gene promoter, enhancer, or repressor) associated with, for example, a gene having a mutation.
  • the method comprises administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition that comprises a polynucleotide encoding a prime editor system (e.g., prime editor and PEgRNA) described herein.
  • a prime editor system e.g., prime editor and PEgRNA
  • the prime editor is a fusion protein that comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain.
  • a cell of the subject is transduced with the prime editor and one or more PEgRNA guide polynucleotides that direct the prime editor to effect a desired nucleotide edit in a disease-associated gene, a disease- causing gene, or a regulatory nucleic acid sequence associated with a disease-causing gene.
  • the desired nucleotide edit effected by prime editing my correct a disease associated mutation in the disease causing gene to a wild type gene sequence.
  • the methods herein include administering to the subject (including a subject identified as being in need of such treatment, or a subject suspected of being at risk of disease and in need of such treatment) an effective amount of a composition described herein. 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).
  • 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).
  • cells are obtained from the subject and contacted with a pharmaceutical composition as provided herein.
  • cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected by the prime editor or detected in the cells.
  • pharmaceutical compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use.
  • prime editors engineered RTs
  • engineered Cas-RTs Also disclosed are engineered RTs and engineered Cas-RTs.
  • the engineered RTs and the engineered Cas-RTs may comprise amino acid or sequence variations of naturally occurring RTs and Cas-RTs.
  • the prime editors, engineered RTs, or engineered Cas-RTs may also comprise heterologous functional domains comprising SET domains. Other functional domains may comprise nuclear localization signals or sequences or linkers.
  • the prime editors, engineered RTs, or engineered Cas-RTs may complex with PEgRNAs.
  • prime editing may comprise the prime editors, engineered RTs, engineered Cas-RTs, or PEgRNAs.
  • a cell may comprise the prime WSGR Docket No.59761-781.601 editors, engineered RTs, engineered Cas-RTs, PEgRNAs, any derivatives thereof, or any combinations thereof.
  • a composition may comprise the prime editors, engineered RTs, engineered Cas-RTs, PEgRNAs, any derivatives thereof, any combinations thereof, or the cells comprising thereof.
  • a method for installing a nucleotide edit may comprise the prime editors, engineered RTs, engineered Cas-RTs, or PEgRNAs.
  • a method for treating a disorder may comprise the prime editors, engineered RTs, engineered Cas-RTs, PEgRNAs, any derivatives thereof, any combinations thereof cells comprising thereof, compositions comprising thereof, or kits comprising thereof.
  • Prime editing, the methods for installing a nucleotide edit, the methods for treating a disorder, the methods of reverse transcribing an RNA sequence using the prime editors, engineered RTs, engineered Cas-RTs, PEgRNAs, any derivatives thereof, or any combinations thereof cells comprising thereof are also illustrated in the examples described herein. [0460] 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 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.
  • components of a prime editor composition may be delivered as a combination of DNA and RNA.
  • components of a prime editor composition can be delivered as a combination of nucleic acid, e.g., DNA and/or RNA and protein.
  • a prime editing composition component is encoded by a polynucleotide, a vector, or a construct.
  • a prime editor polypeptide, a PEgRNA and/or a ngRNA 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.
  • the polynucleotide encodes a DNA polymerase domain of a prime editor.
  • the polynucleotide encodes a portion of a prime editor protein, for example, a N- 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. In some embodiments, the polynucleotide encodes a PEgRNA and/or a ngRNA. In some embodiments, 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 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 WSGR Docket No.59761-781.601 a 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. may be used in the expression vector (e.g., U6 promoter, H1 promoter).
  • the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector, e.g., a plasmid or a virus.
  • the vector is a viral vector.
  • the vector is a non-viral vector.
  • delivery is in vivo, in vitro, ex vivo, or in situ.
  • Non-viral vector delivery systems may include DNA plasmids, RNA (e.g., a transcript of a vector described herein), virosome, viral like particle, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • the polynucleotide is provided as an RNA, e.g., an 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.
  • an 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, PEgRNA, and/or ngRNA 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 gene or can be introduced into a cell using any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection).
  • the prime editor-coding sequences, the PEgRNAs, and/or the ngRNAs 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, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, 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.
  • Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, can be used.
  • the compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes may be anionic, neutral or cationic.
  • 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 (ex vivo).
  • the virus is selected from a group I virus (e.g., a dsDNA virus), group II virus (e.g., a ssDNA virus), group III virus (e.g., a dsRNA virus), group IV virus (e.g., a +ssRNA virus), group V virus (e.g., a -ssRNA virus), group VI virus (e.g., a ssRNA-RT virus), or a group VII virus (e.g., a dsDNA-RT virus).
  • 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.
  • the AAV is a recombinant AAV (rAAV).
  • 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 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 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.
  • 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 is fused to an intein.
  • the portion or fragment of the polypeptide can be fused to the N-terminus or the C-terminus of the intein.
  • a 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.
  • the 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 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.
  • intein refers to a self- splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (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 Cas12i2.
  • 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 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 including 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: 936).
  • 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 (SEQ ID NO: 937), nona-arginine, and octa-arginine (SEQ ID NO: 938).
  • the nona-arginine (R9) sequence (SEQ ID NO: 937) 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/ngRNA are introduced to a target cell by nanoparticles.
  • the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle.
  • any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components.
  • the nanoparticle is inorganic.
  • the nanoparticle is organic.
  • a prime editing composition is delivered to a target cell, e.g., a hepatocyte, in an organic nanoparticle, e.g., a lipid nanoparticle (LNP), (e.g., a cationic lipid nanoparticle, an ionizable lipid nanoparticle), a micelle, polymer nanoparticle, Lipid—polymer nanoparticles (PLNs), or a combination thereof.
  • LNP lipid nanoparticle
  • PPNs Lipid—polymer nanoparticles
  • LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof.
  • neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability.
  • a lipid nanoparticle may comprise a conjugated lipid, e.g., a PEG-phospholipid.
  • Lipid nanoparticles may include additional elements, e.g., a polymer.
  • 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.
  • the lipid particle comprises a cationic lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and/or a sterol.
  • exemplary lipids used to produce LNPs are provided in Tables 20 and 21 below.
  • a cationic lipid may be an ionizable cationic lipid, e.g., a cationic lipid that may carry a positive charge or be neutral depending on pH, or an amine- containing lipid that can be readily protonated.
  • a lipid nanoparticle may comprise a second cationic lipid.
  • a polynucleotide encoding a prime editor polypeptide component may be co-formulated with a cationic lipid.
  • the nucleic acid may be encapsulated in an LNP.
  • the LNP formulation is biodegradable.
  • LNPs are directed to specific tissues e.g., by displaying biological ligands on the surface of LNPs to enhance interaction with cognate receptors.
  • all components of a prime editor may be delivered in a single LNP formulation. In some embodiments, components of a prime editor may be delivered by separate LNP formulations.
  • components of a prime editing composition form a complex prior to delivery to a target cell.
  • a prime editor fusion protein, a PEgRNA, and/or a ngRNA 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 or ngRNA
  • RNP ribonucleoprotein
  • 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 20 and 21 below.
  • Table 20 Exemplary lipids for nanoparticle formulation or gene transfer WSGR Docket No.59761-781.601
  • Exemplary polymers for use in nanoparticle formulations and/or gene transfer are shown in Table 21 below.
  • Table 21 Exemplary lipids for nanoparticle formulation or gene transfer
  • Exemplary delivery methods for polynucleotides encoding prime editing composition components are shown in Table 22 below.
  • the prime editing compositions of the disclosure may be provided to the cells 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 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 WSGR Docket No.59761-781.601 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.
  • two or more different prime editing system components e.g., two different polynucleotide constructs are administered to the subject (e.g., different components of the same prim 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.
  • pegRNA and ngRNA assembly For each pegRNA or nicking guide RNA (ngRNA), oligos encoding a spacer, a gRNA scaffold, and an extension arm (for nicking guide RNA, a spacer and a gRNA scaffold) are ligated by Gibson assembly or Golden Gate assembly and cloned into a U6 expression plasmid as described in Anzalone et al., Nature.2019 Dec; 576(7785): 149-157.
  • ngRNA nicking guide RNA
  • HEK293T cells are seeded on 48-well poly-D-lysine coated plates (Corn- ing). Between 16 and 24 h after seeding, cells are transfected at approximately 60% confluency with 1 ⁇ l lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocols and 750 ng plasmid that expressed prime editors and 250 ng plasmid that expressed PEgRNAs. Cells are cultured for 3 days following transfection, after which the medium was removed, the cells were washed with 1 ⁇ PBS solution (Thermo Fisher Scientific).
  • Genomic DNA is extracted by the addition of 150 ⁇ l of freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.5; 0.05% SDS; 25 ⁇ g/ml proteinase K (ThermoFisher Scientific) directly into each well of the tissue culture plate. [0491] –2 h, i are generated, barcoded, and sequenced on a Miseq from Illumina. Percent editing at the target locus is determined with Crispresso2 (Clement, K. et al., “CRISPResso2 provides accurate and rapid genome editing sequence analysis.” 2019, Nat Biotechnol 37, 224–226).
  • EXAMPLE 2 Installing A Desired Nucleotide Edit In A Cell Using Prime Editing Using Expression Vectors
  • a populations of host cells is transfected with a first vector that expresses a prime editor in the host cell and a second vector that expresses PEgRNA in the host cell.
  • the PEgRNA has a nucleotide edit and a spacer sequence that bound to a pre-determined genomic location.
  • One-week post-transfection the population of host cells successfully transfected with the first and second vector are selected and clonally expanded.
  • the individual host cell clone (targeting HEK3) is tested for being installed the nucleotide edit at the pre-determined genomic location using methods described in EXAMPLE 1.
  • the high throughput sequencing step can also be replaced with Sanger Sequencing of the pre-determined genomic location.
  • EXAMPLE 3 Installing A Desired Nucleotide Edit In A Cell Using Prime Editing Using a Purified Prime Editor [0493] A population of host cells is transfected or electroporated with an mRNA encoding the prime editor and a PEgRNA synthesized ex vivo. The PEgRNA has a nucleotide edit and a spacer sequence that bound to a pre-determined genomic location. One-week post-transfection, the population of host cells WSGR Docket No.59761-781.601 successfully transfected with the first and second vector are selected and clonally expanded.
  • EXAMPLE 4 Installing A Desired Nucleotide Edit In A Cell Using Prime Editing Using Purified RNA [0494] A population of host cells is transfected or electroporated with an mRNA encoding the prime editor and a PEgRNA synthesized ex vivo. The PEgRNA has a nucleotide edit and a spacer sequence that bound to a pre-determined genomic location.
  • Amino acid sequences of exemplary Reverse Transcriptase Homologs (RT domains) WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-
  • Amino acid sequences of exemplary Reverse Transcriptases WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 Table 3.
  • Amino acid sequences of exemplary ASR RT domains. N-terminal methionines are omitted WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 Table 4.
  • Amino acid sequences of exemplary Cas12i proteins WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 Table 5.
  • NLS nuclear localization signals
  • SenR Variant Reverse Transcriptase Sequences for SenR variants, amino acid substitutions indicated are relative to SEQ ID NO: 1125
  • WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601
  • WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601
  • WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601
  • Exemplary SenR112 Variant Reverse Transcriptase (amino acid substitutions relative to SEQ ID NO: 1096) WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 Table 18.
  • Exemplary SenR121 Variant Reverse Transcriptase (amino acid substitutions relative to SEQ ID NO: 1097) WSGR Docket No.59761-781.601 WSGR Docket No.59761-781.601 Table 19.

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Abstract

Des éditeurs primaires pour la réécriture par matrice d'ARN sont présentement divulgués. Des transcriptases inverses modifiées et des méthodes d'utilisation de celles-ci pour la réécriture par matrice d'ARN sont également divulguées.
PCT/US2024/016758 2023-02-22 2024-02-21 Procédés et compositions pour la réécriture de séquences nucléotidiques Ceased WO2024178144A1 (fr)

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* Cited by examiner, † Cited by third party
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CN115698278A (zh) * 2020-03-31 2023-02-03 阿伯生物技术公司 包含Cas12i2变体多肽的组合物及其用途
CN119685290A (zh) * 2023-12-22 2025-03-25 山东舜丰生物科技有限公司 一种用于引导编辑技术的组合物和方法

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WO2022150790A2 (fr) * 2021-01-11 2022-07-14 The Broad Institute, Inc. Variants d'éditeur primaire, constructions et procédés pour améliorer l'efficacité et la précision d'une édition primaire
WO2022212926A1 (fr) * 2021-04-01 2022-10-06 Prime Medicine, Inc. Méthodes et compositions pour l'édition de séquences nucléotidiques
WO2023283092A1 (fr) * 2021-07-06 2023-01-12 Prime Medicine, Inc. Compositions et procédés d'édition de génome efficace
WO2023004439A2 (fr) * 2021-07-23 2023-01-26 Prime Medicine, Inc. Compositions d'édition de génome et méthodes de traitement de maladie granulomateuse chronique

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WO2022150790A2 (fr) * 2021-01-11 2022-07-14 The Broad Institute, Inc. Variants d'éditeur primaire, constructions et procédés pour améliorer l'efficacité et la précision d'une édition primaire
WO2022212926A1 (fr) * 2021-04-01 2022-10-06 Prime Medicine, Inc. Méthodes et compositions pour l'édition de séquences nucléotidiques
WO2023283092A1 (fr) * 2021-07-06 2023-01-12 Prime Medicine, Inc. Compositions et procédés d'édition de génome efficace
WO2023004439A2 (fr) * 2021-07-23 2023-01-26 Prime Medicine, Inc. Compositions d'édition de génome et méthodes de traitement de maladie granulomateuse chronique

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115698278A (zh) * 2020-03-31 2023-02-03 阿伯生物技术公司 包含Cas12i2变体多肽的组合物及其用途
CN115698278B (zh) * 2020-03-31 2025-10-17 阿伯生物技术公司 包含Cas12i2变体多肽的组合物及其用途
CN119685290A (zh) * 2023-12-22 2025-03-25 山东舜丰生物科技有限公司 一种用于引导编辑技术的组合物和方法

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