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WO2024243090A2 - Grandes recombinases de sérine et méthodes d'utilisation - Google Patents

Grandes recombinases de sérine et méthodes d'utilisation Download PDF

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Publication number
WO2024243090A2
WO2024243090A2 PCT/US2024/030106 US2024030106W WO2024243090A2 WO 2024243090 A2 WO2024243090 A2 WO 2024243090A2 US 2024030106 W US2024030106 W US 2024030106W WO 2024243090 A2 WO2024243090 A2 WO 2024243090A2
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Prior art keywords
sequence
seq
rrs
nucleic acid
acid sequence
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WO2024243090A3 (fr
Inventor
Brian R. CHAIKIND
Stepan TYMOSHENKO
Rohan Grover
David PAEZ-ESPINO
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Mammoth Biosciences Inc
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Mammoth Biosciences Inc
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Publication of WO2024243090A2 publication Critical patent/WO2024243090A2/fr
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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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • LSRs Large serine recombinases
  • integrases catalyze the movement of DNA elements into and out of bacterial chromosomes using site-specific recombination between short DNA "attachment sites”.
  • the LSRs that function as bacteriophage integrases cany' out integration between attachment sites in the phage (attP) and in the host (attB).
  • Site-specific recombinases such as large serine recombinases, have evolved to catalyze the transfer of large genetic payloads that are often tens of kilobases in length, from one organism to another, without relying on recipient genetic repair machinery.
  • recombinases are capable of catalyzing target cleavage, strand exchange and DNA rejoining within their synaptic complexes. This mechanism enables site-specific DNA insertion without requiring any cellular cofactors and without generating exposed DSBs.
  • LSRs do not have to be engineered to prevent excision of the integration product once integration occurs.
  • An LSR is usually 400-700 amino acids in length including a catalytic site and several domains that are used for sequence recognition and placement for the integration to occur.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas proteins Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR/Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequencespecific manner
  • Native systems contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets.
  • LSRs large serine recombinases
  • the disclosure provides systems and methods for using CRISPR systems to generate attachment sites where integration of a donor DNA via an LSR is desired.
  • the present disclosure provides a system for the modification of a target nucleic acid comprising: a recombinase or a nucleic acid encoding a recombinase; and a donor DNA vector comprising a first recombinase recognition sequence (RRS-1 ).
  • the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761-9763
  • the target nucleic acid comprises a second RRS (RRS-2).
  • the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2523 and 9761 -9763
  • the RRS-1 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs:6442-8964, 9740-9760, and 9764-9791
  • the RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherem the recombinas
  • the RRS-l and/or RRS-2 is located in or near a safe harbor locus
  • the safe harbor locus is near an AAPS1 (PPPIR12C) gene, an ALB gene, an ANGPTL3 gene, an APOC3 gene, an ASGR2 gene, a CCR5 gene, a FIX (F9) gene, a G6PC gene, a GYS2 gene, an HGD gene, a LP(A) gene, a PCSK9 gene, a SERF INA I gene, a TF gene, and a TTR gene, and an intron thereof.
  • AAPS1 PPIR12C
  • ALB ALB gene
  • ANGPTL3 gene an APOC3 gene
  • ASGR2 gene a CCR5 gene
  • FIX (F9) gene a G6PC gene
  • GYS2 gene a GYS2 gene
  • HGD gene a LP(A) gene
  • PCSK9 gene a SERF INA I gene
  • the RRS-1 and/or RRS-2 is located in or near a target sequence of the human albumin gene (ALB) or AA VS1 gene.
  • the target sequence is selected from a nucleotide sequence that is at least 80%, at least 90%. at least 95% or 100% identical to a sequence selected from SEQ ID NGs: 8965-9266, and wherein the target sequence is in the same row of TABLE 1 as the recombinase. RRS-1, and RRS-2.
  • the donor DNA vector is a circularized DNA molecule.
  • the recombinase or nucleic acid encoding the recombinase, and the donor DNA vector are combined in a single composition.
  • the nucleic acid encoding the recombinase comprises the donor DNA.
  • the present disclosure provides methods of modifying a target nucleic acid comprising contacting the target nucleic acid with the components of a composition or system described herein.
  • the present disclosure provides methods of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the components of a composition or system described herein [0016]
  • the present disclosure provides compositions for the modification of a target nucleic acid, wherein the compositions comprise a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises: a recombinase comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1 - 3918 and 9761-9763; an effector protein; and an RNA dependent DNA polymerase (RDDP).
  • RDDP RNA dependent DNA polymerase
  • the effector protein is a CRISPR associated (C ’as) protein.
  • the Cas protein is a Type V Cas protein.
  • the Cas protein comprises an amino acid sequence that is at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs. 9267-9401 and 9698-9701 .
  • the length of the Cas protein is 350 to 550 amino acids. In some embodiments, the length of the Cas protein is 550 to 800 amino acids.
  • the Cas protein comprises nickase activity.
  • the RDDP comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.
  • the compositions comprise an extended guide nucleic acid, wherein the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridises to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the second strand of the target nucleic acid; and iv) a template sequence that hybridizes to a second portion of the second strand of the target nucleic acid.
  • the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridises to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion
  • the composition comprises a first guide nucleic acid
  • the extended guide nucleic acid comprises: i) a protein binding sequence, wherein the effector protein is capable of binding the protein binding sequence, ii) a spacer sequence that hybridizes to a first target sequence on a first strand of the target nucleic acid, iii) a primer binding sequence, optionally wherein a portion of the primer binding sequence hybridizes to a first portion of the first strand of the target nucleic acid; and iv) a template sequence that hybridizes to a second portion of the first strand of the target nucleic acid.
  • compositions comprise a donor DNA vector, wherein the donor DNA vector first RRS (RRS-1).
  • the template sequence of the extended guide nucleic acid comprises at least a portion of a first or second RRS, and the target sequence optionally cotriprises a portion of the first or second RRS.
  • the composition comprises a second guide nucleic acid, wherein the guide nucleic acid comprises a protein binding sequence and a spacer sequence that hybridizes to a second target sequence on the second strand of the target nucleic acid.
  • the fusion protein comprises a nuclear localization signal.
  • the present disclosure provides methods of modifying a target nucleic acid comprising contacting the target nucleic acid with a composition provided herein. In some embodiments, the present disclosure provides methods of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the components of a system of described herein.
  • a target nucleic acid comprising: (a) a recombinase or a nucleic acid encoding a recombinase; (b) a donor DNA vector comprising a first recombinase recognition sequence (RRS-1). wherein the target nucleic acid comprises a second RRS (RRS-2); and wherein
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least
  • the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6444 or 9766; and the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3921 or 9794;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 16; the R.RS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%.
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3934, 9719, or 9792;
  • the recombinase comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6459, 9743, or 9767:
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3936, 9722, or 9795;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 20;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6461 , 9741 , or 9765;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3938. 9720, or 9793;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 350;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6791, 9756, or 9780;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4268, 9735, or 9808;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 441;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6882, 9757, 9781, or 9784 ;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%. at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4359, 9736. 9809, or 9812;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 454;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6895, 9751 , or 9775;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4372, 9730, or 9803;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 455;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6896, 9750, or 9774;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEC) ID NOs: 4373. 9729, or 9802;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 569;
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7010, 9748, or 911T.
  • RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4487, 9727, or 9800;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 591: the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%.
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4509, 9734, or 9807;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 020:
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7061 , 9758, or 9782:
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 4538, 9737, or 9810;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 955:
  • the RRS-1 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7396 or 9791 ;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of SEQ ID NOs: 4873 or 9819;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 105;
  • the RRS-1 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7546, 9760, or 9783;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5023, 9739, or 9811;
  • the recombinase comprises or consists of an amino acid sequence that is at ieast 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1112;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7553, 9754, or 9778;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5030, 9733, or 9806;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1162;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%.
  • the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5080 or 9817;
  • the recombinase comprises or consists of an amino acid sequence that is at ieast 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1319;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7760, 9745, or 9769;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at ieast 85%, at least 90%, at ieast 95%, at ieast 98%, at ieast 99%, or 100% identical to any one of SEQ ID NOs: 5237. 9724, or 9797;
  • the recombinase comprises or consists of an amino acid sequence that is al least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1452;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7893, 9749, or 9773;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5370, 9728, or 9801;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 1518;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at feast 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7959, 9752, or 9776;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5436, 973 I , or 9804;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1735;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%. at least 99%.
  • the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of SEQ ID NOs: 5653 or 9816;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1944;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8385, 9746, or 9770;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5862. 9725, or 9798;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1966;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8407, 9753, or 9777;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5884, 9732, or 9805;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, al least 75%, at least 80%, at least 85%, at least 90%, at least 95%, al least 98%, at least 99%. or 100% identical to SEQ ID NO: 2050;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at feast 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8491, 9744, or 9768;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5968, 9723, or 9796;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2068;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8509, 9759, or 9790;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5986, 9738, or 9818;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 2172;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 8613, 9747, or 9771;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 6090. 9726, or 9799;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%. at least 85%>, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9761 ;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9785;
  • the RRS-2. comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9813;
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 9762:
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9786;
  • the RRS-2 comprises or consists of an amino acid sequence that is at least 70%. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 98%. at least 99%. or 100% identical to SEQ ID NO: 9814: or
  • the recombinase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9763;
  • the RRS-l comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9787;
  • the RRS-2 comprises or consists of an ammo acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9815.
  • a system for the modification of a target nucleic acid comprising (a) a recombinase or a nucleic acid encoding a recombinase; (b) a donor DNA vector comprising a first recombinase recognition sequence (RRS-l), wherein the target nucleic acid comprises a second RRS (RRS-2), wherein the recombinase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a protein any one of SEQ ID of column 3 of TABLE 16, and (c) wherein at least one of the RRS- 1 and RRS-2 comprises a corresponding kmer sequence of TABLE 16; wherein in the protein and the kmer are on the same line of TABLE 16,
  • FIG, 1 A - FIG. IC illustrate examples of DNA rearrangement mediated by large serine recombinases and recombination directionality factors (RDFs).
  • FIG. 1A shows DNA integration mediated by RRS-l K RRS-2 + LSR recombination.
  • FIG. IB shows recombination
  • FIG. 1C shows DNA inversion through RRS-1 - ⁇ RRS-2 recombination.
  • FIG. 2A - FIG. 2C illustrate exemplary methods on modifying a target nucleic acid sequence with the large serine recombinase systems described herein.
  • FIG. 2A illustrates methods utilizing one pair of RRS-1 x RRS-2 sites.
  • FIG. 2B illustrates methods utilizing two pairs of RRS-1 x RRS-2 sites and one LSR.
  • FIG. 2C illustrates methods utilizing two pairs of RRS-1 x RRS-2 sites and two LSRs.
  • FIG. 2A - FIG. 2C illustrates exemplary methods of target nucleic acid modification, wherein the donor nucleic acid is present as a circular RNA.
  • FIG. 3 shows an exemplary gene editing system comprising an extended guide RNA with the primer binding sequence and template sequence located 5' of a repeat sequence and a spacer sequence.
  • the effector protein is fused to the RNA-directed DNA polymerase (RDDP).
  • RDDP RNA-directed DNA polymerase
  • System components shown in FIG. 3 are exemplary and non-limiting.
  • the 5’ extended rtgRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.
  • FIG. 4 shows an exemplary gene editing system comprising an extended guide RNA with the primer binding sequence and template sequence located 3’ of a repeat sequence and a spacer sequence.
  • the effector protein is fused to the RNA-directed DNA polymerase (RDDP), System components shown in FIG. 4 are exemplar ⁇ and non-limiting.
  • the 5’ extended rtgRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.
  • FIGs. 5A-5B show exemplary split protein RNA design gene editing systems comprising a guide RN A with the repeat and spacer sequences separate from the primer binding sequence and template sequence.
  • the effector protein is separate from the RDDP
  • the effector protein is localized to the target nucleic acid via the guide RNA where it can nick the target nucleic acid.
  • the primer biding sequence and template sequence are linked to an MS2 aptamer.
  • the RDDP is fused to an MS2 coat protein that is capable of binding the MS2 aptamer, thereby localizing the RDDP to the nicked DNA.
  • System components shown in FIGs. 5A and 5B are exemplary and non-limiting.
  • the gRNA and/or retRNA may comprise additional nucleotides beyond those labeled as spacer, repeat, linker, PBS and RT template.
  • FIG. 6 shows an overview of a system comprising CasM.265466 for precise editing.
  • CasM.265466 generates a double-stranded break on target DNA.
  • a retRNA hybridizes with the non-target strand (as shown) or target strand (not shown) via complementarity between the PBS region and the target DNA.
  • An RDDP is recruited to the target site by binding of a fused MS2 coat protein to the MS2 aptamer of the retRNA.
  • the RNA/DNA hybrid serves as a binding substrate for the RDDP. which then synthesizes new cDNA along the exposed 3" end of the target DNA using the RT template as a reverse transcription template. Edits encoded in the RT template are thereby written into the genome.
  • FIG. 7 shows incorporation of a donor DNA reporter (mCherry) into safe harbor loci (albumin and A4FS7) of mammalian cells by LSRs disclosed herein.
  • FIG. 8 shows a representative diagram of a promoter swap assay via DNA inversion. as also exemplified in FIG. 1C.
  • FIG, 9 shows a representative diagram of a reporter swap assay via DNA inversion, as also exemplified in FIG. IC.
  • FIG. 10 shows the results of a promoter swap assay demonstrating that the LSR proteins have recombinase activity in mammalian cells.
  • FIG. 11 show's the results of a reporter swap assay demonstrating that the LSR proteins have recombinase activity in mammalian cells.
  • FIG. 12 shows the results of a reporter swap assay demonstrating that the LSR proteins can integrate large payloads into a gene sequence.
  • FIG. 13A-FIG. 13D show' the results of additional LSR proteins used in the reporter swap assay.
  • FIG. 13A and FIG. 13C show 7 the results of the reporter swap assay as a percent of mCherry expression, which demonstrates recombination efficiency.
  • FIG. 13B and FIG. 13D show the percent GFP expression, which demonstrates the overall transfection efficiency.
  • FIG. 14A-FIG. 14B show a representative assay (FIG. 14A) to test for the integration efficiency of a donor plasmid comprising an atB or attP site and the results (FIG. 14B) of said assay.
  • % identical refers to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity.
  • the % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100.
  • computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.
  • sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/nisa/muscle/, mafft.cbrc.jp/alieiiment/software/. See, e.g., Altschul et al. (1990). J. Mol. Bioi. 215:403-10.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural. or derivatized nucleotide bases.
  • Binding refers to a non-covalent interaction between macromolecules (e.g.. between a protein and a nucleic acid; between a CAS polypeptide/guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions are generally characterized by a dissociation constant (KD) of less than 10" 6 M, less than 10’ 7 M, less than 1O‘ S M, less than 10' 9 M, less than 10' ,y M, less than IO' 11 M, less than 10' 12 M, less than IO’ 13 M, less than I O' 14 M. or less than I O' 15 M.
  • KD dissociation constant
  • Affinity refers to the strength of binding, increased binding affinity being correlated with a lower KD.
  • binding domain it is meant a protein or nucleic acid domain that is able to bind non-covalently to another molecule.
  • a binding domain can bind to, for example, a DNA molecule (a DNA-binding domain), an RNA molecule (an RNA-binding domain) and/or a protein molecule (a protein-binding domain).
  • a DNA-binding domain a DNA-binding domain
  • RNA-binding domain an RNA molecule
  • protein-binding domain a protein-binding domain
  • it can in some cases bind to itself (to form honiodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.
  • cleavage refers to cleavage (hydrolysis of a phosphodi ester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur wi thin or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.
  • nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond.
  • the result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydroly sis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e g.. ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.
  • a nick hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule
  • single strand break hydroly sis of a single phosphodiester bond on a single-stranded molecule
  • double strand break hydrolysis of two phosphodiester bonds on both sides of a double-strande
  • complementary' and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid.
  • C with G Watson-Crick counterparts
  • a with T Watson-Crick counterparts
  • the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3'-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.
  • the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ’reverse complement' sequence or the ’reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3'-end.
  • Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
  • “conservative substitution” as used herein refers to the replacement of one amino acid for another such that the replacement takes place within a family of ammo acids that are related in their side chains.
  • “non-conservative substitution” as used herein refers to the replacement, of one ammo acid residue for another that does not have a related side chain.
  • Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (G): (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Tip (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i ) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T).
  • Ammo acids may be related by aliphatic, side chains: Gly (G). Ala (A), Vai (V), Leu (L). lie (1), Ser (S). Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-bydroxyl.
  • Amino acids may be related by aromatic side chains: Phe (F). Tyr ( ⁇ ), Trp (W).
  • Ammo acids may be related by amide side chains: Asn (N), Glu (Q).
  • Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).
  • donor nucleic acid refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.
  • target nucleic acid refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein.
  • a target nucleic acid may comprise RNA, DNA, or a combination thereof.
  • a target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded
  • target sequence refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to a respective length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
  • a nucleotide sequence that “encodes” a particular polypeptide or protein is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and/or is translated (in the case of mRNA) into a polypeptide.
  • transgene refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell .
  • a transgene is meant to include (1 ) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleoli de sequence that serves to add additional copies of the same (e.g..
  • a donor nucleic acid can comprise a transgene.
  • the cell in which transgene expression occurs can be a target cell, such as a host cell.
  • a functional fragment refers to a fragment of a protein that retains some function relative to the entire protein.
  • functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity.
  • a functional fragment may be a recognized functional domain, e.g., a catalytic domain such as, but not limited to, a RuvC domain.
  • fusion effector protein may be used interchangeably herein and refer to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein.
  • fusion partner protein or “‘fusion partner,” as used herein, refer to a protein, polypeptide or peptide that is fused, or linked via a linker, to an effector protein.
  • the fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein.
  • effector protein refers to a polypeptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid.
  • a complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein
  • the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid
  • the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid.
  • a non-limiting example of an effector protein modify ing a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout.
  • reference to an effector protein includes reference to a nucleic acid encoding the effector protein, unless indicated otherwise.
  • genetic disease refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.
  • guide nucleic acid refers to at least one nucleic acid comprising: a first nucleotide sequence that complexes to an effector protein on either the 5’ or 3’ terminus and the first nucleotide sequence can be fused to a second nucleotide sequence that hybridizes to a target nucleic acid.
  • the first sequence may be referred to herein as a repeat sequence or guide sequence.
  • the second sequence may be referred to herein as a spacer sequence.
  • guide nucleic acid may be referred to interchangeably with the term, “guide RNA, ” It is understood that guide nucleic acids may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone.
  • extended guide RNAs may comprise DN A, RNA, or a combination thereof (e.g., RNA with a thymine base).
  • template RNA refers to anucleic acid comprising: a primer binding sequence and a template sequence. It is understood that template RNAs may comprise DNA, RNA. or a combination thereof (e.g., RNA with a thymine base). In some embodiments, the template RNA is linked to a guide RNA via a linker sequence to form an rtgRNA.
  • template sequence and “RT template” as used herein, ref ers to a portion of a retRNA that contains a desired nucleotide modification relative to a target sequence or portion thereof.
  • the desired edit may comprise one or more nucleotide insertions, deletions or substitutions relative to a target sequence or portion thereof. In some embodiments, it is identical to, complementary to, or reverse complementary to a target sequence or portion thereof.
  • the template sequence is complementary' to a sequence of the target nucleic acid that is adjacent to a nick site of a target site to be edited, with the exception that it includes a desired edit
  • the template sequence (also referred in some embodiments as the RT template) can be complementary' to at least a portion of the target sequence with the exception of at least one nucleotide.
  • extension refers to additional nucleotides added to a nucleic acid, RNA, or DNA, or additional amino acids added to a peptide, polypeptide, or protein. Extensions may be processed during the formation of the guide RNA. In some embodiments, the extension comprises or consists of a template RNA. [0078] By “hybridizable” or “complementary " or “substantially complementary” it is meant that a nucleic acid (e.g.
  • RNA, DNA comprises a sequence of nucleotides that enables it to noncovalently bind, i.e, form Watson-Crick base pairs and/or G/U base pairs, "anneal", or “hybridize.” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • Standard Watson-Crick basepairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C).
  • guanine (G) can also base pair with uracil (U).
  • G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon basepairing with codons m mRNA.
  • a guanine (G) e.g...
  • dsRNA duplex of a guide RNA molecule is considered complementary' to both a uracil (U) and to an adenine (A).
  • U uracil
  • A adenine
  • a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary'.
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences
  • Tm melting temperature
  • the position of mismatches can become important (see Sambrook et al..
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (eg., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more. 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution, salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.
  • hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically’ hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc,).
  • a polynucleotide can comprise 60% or more, 65% or more. 70% or more, 75% or more, 80% or more. 85% or more, 90% or more, 95% or more, 98% or more, 99% or more.
  • nucleic acid sequence to which it will hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary 7 nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
  • Example methods include BLAST programs (basic local alignment search tools); BioPython (Cock et al (2009) Biopython: freely 7 available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 25, 1422-1423); and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park. Madison Wis.), e.g.. using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482-489), and the like. Unless otherwise noted, the % complementarity of two sequences referenced herein is determined by the BLAST or BioPython programs.
  • heterologous means that the two different polypeptide or polynucleotide sequences are not found similarly connected to one another in a native nucleic acid or protein.
  • a protein that is heterologous to the effector protein is a protein that is not co valently linked via an amide bond to the effector protein in nature.
  • a heterologous protein is not encoded by a species that encodes the effector protein.
  • a guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature.
  • the first sequence is considered to be heterologous with the second sequence
  • the guide nucleic acid may be referred to as a heterologous guide nucleic acid.
  • linked refers to any covalent mechanism by which two amino acid sequences or nucleic acid sequences are connected to each other in sequence.
  • two sequences are linked directly together by a covalent bond (e g., an amide bond or phosphodi ester bond).
  • two sequences are linked together by a peptide or nucleic acid linker.
  • linked amino acids refers to at least two amino acids linked by an amide bond or a peptide bond.
  • linker refers to an amino acid sequence or nucleic acid sequence that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid.
  • modified target nucleic acid refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an I., SR and/or effector protein.
  • the modification is an alteration in the sequence of the target nucleic acid.
  • the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target, nucleic acid.
  • peptide polypeptide
  • protein are used interchangeably herein, refer to a polymeric form of amino acids.
  • a polypeptide may include coded and non-coded ammo acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more sequence modifications, or both.
  • a peptide generally has a length of 100 or fewer linked amino acids.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences.
  • Sequence identity can be determined in a number of different ways. Percent identity’ can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Alischul et al., J. Mol. Biol., 1990, 215, 403-410: Zhang and Madden, Genome Res.. 1997, 7, 649-656). the Gap program (Wisconsin Sequence Analysis Package. Version 8 for Unix, Genetics Computer Group,
  • a DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA.
  • a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may' encode an RNA that is not translated into protein (e.g. tRNA, rRNA.
  • microRNA miRNA
  • ncRNA non-coding RNA
  • guide RNA etc.
  • a "protein coding sequence” or a sequence that encodes a particular protein or polypeptide is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory? sequences
  • DNA regulatory' sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., RNA-guided endonuclease and the like) and/or regulate translation of an encoded polypeptide.
  • a non-coding sequence e.g., guide RNA
  • a coding sequence e.g., RNA-guided endonuclease and the like
  • the terra, “promoter”’ or “promoter sequence” is a DNA regulatory 7 region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.
  • a PAM is required for a complex of an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid.
  • the complex does not require a PAM to modify the target nucleic acid.
  • a PAM sequences is 5’-NTTN-3’, where N can be any nucleic acid.
  • RDDP RNA-dependent DNA polymerase
  • RuvC domain refers to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain embodiments, of processing a pre- crRNA. In some embodiments, the RuvC domain is located near the C -terminus of the effector protein A single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, aRuvCII subdomain and a RuvCIII subdomain.
  • the term “RuvC” domain can also refer to a “RuvC-like” domain.
  • Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (www.ebi.ac.uk/interpro/).
  • a RuvC- like domain may be a domain which shares homology with a region of TnpB proteins of the
  • nickase refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid. A nickase cleaves a phosphodiester bond between two nucleotides of only one strand of dsDNA.
  • the terms, “’nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage.
  • nuclease activity is used to refer to catalytic activ ity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).
  • nucleic acid cleavage e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.
  • wild type as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature is naturally occurring.
  • non-naturally occurring’ and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man or machine.
  • nucleic acid when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or ammo acid, refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule
  • composition or system described herein refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system.
  • a composition may include an effector protein and a guide nucleic acid that do not naturally occur together.
  • an effector protein or guide nucleic acid that is “natural,” “naturally- occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man or machine.
  • variant is intended to mean a form or version of a protein that differs from the wild-type protein A variant may have a different function or activity relative to the wild- type protein.
  • nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro-transcription, cloning, enzymatic, or chemical cleavage, etc.
  • the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions within the nucleic acid.
  • nuclear localization signal refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.
  • nucleotide(s)’ and/or “nucleoside(s)”. in the context of a nucleic acid molecule having multiple residues, is interchangeable and describe the sugar and base of the residue contained in the nucleic acid molecule.
  • nucleic acid molecule having multiple residues is interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule.
  • nucleobase(s) When referring to a “nucleobase(s)’; or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides.
  • nucleotides, nucleosides, and/or nucieobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity' or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement).
  • nucleoside analogs such as modified uridines
  • sequence 5’-AXG-3' where X is any modified uridine, such as pseudouridine, N1 -methyl pseudo uridine, or 5-methoxyuridine is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5 ! -CAU-3’).
  • the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not. naturally occur. Instead, a. "recombinant" polypeptide is encoded by a recombinant non-naturally occurring DMA sequence, but the amino acid sequence of the polypeptide can be naturally occurring ("wild type") or non-naturally occurring (e.g., a variant, a mutant, etc. ).
  • a recombinant polypeptide is the product of a process run by a human or machine.
  • a "vector” or “expression vector” is a nucleic acid replicon, such as plasmid, phage, virus, artificial chromosome, or cosmid, to which another nucleic acid segment, i.e., an "insert", may be atached so as to bring about the replication of the attached segment in a cell.
  • the term “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle.
  • An "expression cassette” comprises a DNA coding sequence operably linked to a promoter.
  • "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner
  • a promoter is operably linked to a coding sequence (or the coding sequence can also be said to be operably linked to the promoter) if the promoter affects its transcription or expression.
  • Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the inserts) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • a cell has been "genetically modified,” “transformed,” or “transfected” by exogenous DNA or exogenous RNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell.
  • exogenous DNA e.g., a recombinant expression vector
  • the presence of the exogenous DNA results in permanent or transient genetic change.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the ceil. In prokaryotes, yeast, and mammalian ceils for example, the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter ceils through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations
  • Suitable methods of genetic modification include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEl)-mediated transfection, DEAE-dextran mediated transfection. liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g.. Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169- 409X(l 2)00283-9. doi: 10. 1016/j.addr 2012 09.023), and the like.
  • transformation include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEl)-mediated transfection, DEAE-dextran mediated transfection. liposome-mediated transfection, particle gun technology, calcium
  • Nuclease and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).
  • cleavage domain By “cleavage domain,” “active domain,” or “nuclease domain” of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for nucleic acid cleavage.
  • a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
  • a single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.
  • treatment or ’treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results m the recipient.
  • Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • a '‘syndrome”, as used herein, refers to a group of symptoms which, taken together, characterize a condition.
  • the terms "individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • the subject may be a mammal.
  • the subject may be a human
  • the subject may be diagnosed or at risk for a disease
  • LSR Large Serine Recombinase
  • Recombinase recognition sequence refers to the nucleic acid sequences present in donor and target DNA nucleic acids recognized by an LSR described herein. Recombinase recognition sequences may also be referred to in the art as DNA attachment sites. LSR systems were originally described in the context of bacterial and phage integration systems. Therefore, the RRS in the donor nucleic acid is referred to as “attP” (e.g., phage attachment site, used interchangeably with “RRS-i”) and the RRS in the target nucleic acid is referred to as “atB” (e.g , bacterial attachment site, used interchangeably with “RRS-2”).
  • attP e.g., phage attachment site, used interchangeably with “RRS-i”
  • atB e.g , bacterial attachment site, used interchangeably with “RRS-2”.
  • the product of attP * attB recombination is the integration of the donor nucleic acid sequence into the target nucleic acid sequences, in which the donor nucleic acid sequence is flanked by two new recombination sites, attL (used interchangeably with “RRS-L”) and attR (used interchangeably with “RRS-R”), each containing half sites derived from attP and attB, See FIG. 1A-FIG. 2C.
  • base editing enzyme refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the chemical modification of a nucleobase of a deoxyribonucleotide or a ribonucleotide.
  • a base editing enzyme for example, is capable of catalyzing a reaction that modifies a nucleobase that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded).
  • Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleobase to a different nucleobase, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e g., CpG, CpA, CpT or CpC).
  • a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase.
  • base editor refers to a fusion protein comprising a base editing enzyme linked to an effector protein.
  • the base editing enzyme may be referred to as a fusion partner.
  • the base editing enzyme can differ from a naturally occurring base editing enzyme. It is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant.
  • the base editor is functional when the effector protein is coupled to a guide nucleic acid.
  • the guide nucleic acid imparts sequence specific activity to the base editor.
  • the effector protein may comprise a catalytically inactive effector protein.
  • the base editing enzyme may comprise deaminase activity
  • LSRs can catalyze recombination between RRS sites on linear or circular DNA substrates and, depending on the position and orientation of the RRS sites, integrate, excise or invert sections of DNA (See examples of DNA editing by LSRs in FIGs. 1A-C).
  • LSRs mediate unidirectional recombination between a first RRS site (RRS- 1) in the donor nucleic acid sequence and a second RRS site (RRS-2) in the target nucleic acid sequence, generating RRS-L (left) and RRS-R (right) sites flanking the 5’ and 3’ ends (respectively) of the integrated donor nucleic acid sequence.
  • LSRs do not degrade DNA or need to be engineered to bind a specific DNA sequence (in contrast to methods that rely on homing endonucleases);
  • integration can be targeted to a specific locus known to have minimal positional effects on transgene expression, unlike the use of transposons and retroviruses; and (d) transgenes integrated by LSRs though RRS-1 * RRS-2 recombination cannot be inverted or remobilized without the presence of a cognate RDF.
  • the present disclosure provides an LSR, or a nucleic acid encoding the same, wherein the LSR comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an ammo acid sequence selected from SEQ ID NOs: 1-3918 and 9761 -9763.
  • the LSR comprises or consists of an amino acid sequence selected from SEQ ID NOs: 1-3918 and 9761-9763.
  • the present disclosure provides methods of modifying a target nucleic acid sequence using an LSR system described herein. Exemplary embodiments of combinations of system elements and methods of modification are described below.
  • the system provided herein comprises the following elements: (a) an LSR or a nucleic acid encoding the same: and (b) a donor DNA vector comprising a first recombinase recognition sequence
  • the target nucleic acid comprises a second recombinase recognition sequence (RRS-2) that may be naturally occurring or may be introduced into the target nucleic acid sequence.
  • the donor DNA vector may be a circular DNA.
  • the entirety of the donor DNA vector can be inserted into the target nucleic acid sequence. See e.g. , FIG. 2A.
  • system comprises the following elements:
  • a donor DNA vector comprising two recombinase recognition sequences (RRS- 1A and RRS-1B) flanking the 5’ and 3’ ends of a sequence of interest (SOI) (or gene of interest
  • the target nucleic acid comprises two recombinase recognition sequences (RRS-2A and RRS-2B) that flank the 5" and 3 r end of the target nucleic acid sequence
  • the donor DNA may be circular DNA.
  • the LSR mediates recombination between RRS-1 A x RRS-2A and RRS-1B x RRS-2B such that a portion of the target nucleic acid sequence is excised and replaced with the sequence of interest from the donor DNA vector See e.g., FIG. 2B.
  • the target nucleic acid comprises two recombinase recognition sequences (RRS-2A and RRS-2B) that flank the 5’ and 3' end of the target nucleic acid sequence.
  • the donor DNA may be circular DNA.
  • the first LSR mediates recombination between RRS-1A x RRS-2A and the second LSR mediates recombination between RRS-1B x RRS-2B such that a portion of the target nucleic acid sequence is excised and replaced with the sequence of interest from the donor DNA vector See e.g. , FIG, 2C.
  • the LSR comprises an ammo acid sequence that is at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2523 and 9761-9763;
  • the RRS- 1 comprises a nucleotide sequence that is at least 60%, at least 70%. at least 80%, at least 85%, at least 90%. at least 95%. or 100% identical to a sequence selected from SEQ ID NOs:6442- 8964.
  • RRS-2 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase.
  • RRS-1 and RRS-2 are in the same row' of TABLE 1 or TABLE 2.
  • the LSR comprises an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441, 454. 455, 569, 591. 620, 955, 1 105, 1112, 1 162, 1319. 1452, 1518, 1735, 1944, 1966, 2050, 2068, 2172, 9761 , 9762. and 9763; the R.RS-1 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%.
  • the RRS-2 comprises a nucleotide sequence that is at least 60%. al least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 9719, 9720, 9722-9739, 9793-9798, 9802-9809, and 9813-9819, wherein the recombinase, RRS-1 and RRS-2 are in the same row of any one of TABLES 13-15.
  • the target sequence is selected from a nucleotide sequence that is at least 80%, at least 90%. at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 8965-9266. and wherein the target sequence is in the same row of TABLE 1 as the recombinase, RRS-1, and RRS-2.
  • the LSR or nucleic acid encoding the same, and a donor DNA vector are combined in a single composition.
  • the nucleic acid encoding the recombinase further comprises the donor DNA or sequence of interest.
  • the present disclosure comprises system for the modification of a target nucleic acid comprising (a) a recombinase or a nucleic acid encoding a recombinase; and (b) a donor DNA comprising a first recombinase recognition sequence (RRS-1), and wherein the target nucleic acid comprises a second RRS (RRS-2)
  • RRS-1 and/or RRS-2 are less than about 250 nucleotides in length. In some embodiments, the RRS-1 and/or RRS-2 are less than about 250, 200, 150, 100, or 50 nucleotides in length. In some embodiments, the RRS-1 and/or RRS-2.
  • the RRS-1 comprises a nucleic acid sequence that is at least
  • the RRS-1 comprises a nucleic acid sequence selected from SEQ ID NOs: 6442- 8964 In some embodiments, the RRS-1 consists of a nucleic acid sequence selected from SEQ ID NOs: 6442-8964, 9740-9760. and 9764-9791 .
  • the RRS-1 comprises a nucleic acid sequence that is at least 60%, al least 70%, al least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791 and the RRS- 2 comprises a nucleotide sequence that is at least 60%. at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 3919- 6441, 9719-9739, and 9792-9819, wherein the RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2.
  • the RRS-1 comprises a nucleic acid sequence selected from SEQ ID NOs: 6442-8964, 9740-9760. and 9764-9791 and the RRS-2 comprises a nucleotide sequence selected from SEQ ID NOs: 3919-6441, 9719-9739. and 9792-9819, wherein the RRS-1 and RRS-2 are in the same row of TABLE 1 or TABLE 2.
  • the RRS-1 consists of a nucleic acid sequence selected from SEQ ID NOs: 6442- 8964.
  • 9740-9760, and 9764-9791 and the RRS-2 consists of a nucleotide sequence selected from SEQ ID NOs: 3919-6441 , 9719-9739, and 9792-9819, wherein the RRS-I and RRS-2 are in the same row of TABLE 1 or TABLE 2.
  • an RRS is naturally occurring in a target nucleic acid sequence.
  • an RRS is introduced into a target nucleic acid sequence. In some embodiments, one or more RRSs are introduced into the target nucleic acid sequence. In some embodiments, only one RRS is introduced into a target nucleic acid sequence. In some embodiments, two RRSs are introduced into a target nucleic acid sequence. In some embodiments, the one or more RRSs are introduced into the target nucleic acid sequence using the systems described herein. In some embodiments, the guide nucleic acids described herein comprise one or more RRSs.
  • an RRS is introduced into a target nucleic acid sequence with a precision editing system described herein. In some embodiments, an RRS is introduced into a target nucleic acid sequence with a method of precision editing described herein. In some embodiments, the precision editing system comprises a retRNA. In some embodiments, the retRNA comprises an RRSs. In some embodiments, the template sequence of the retRNA comprises one or more RRSs.
  • the RRS-1 and/or the RRS-2 sequence comprise a kmer.
  • a “kmer” is a sequence that, for a given LSR, is in each of the RRS-1, RRS-2, RRS-R, and RRS- L sequences, and is the minimal sequence required for the recombinase uses to mediate DNA rearrangements.
  • a kmer for a given I., SR can be identified by querying the genomic DNA near the prophage boundary 1 within 500 bp of the recombinase, identifying the largest duplicate sequence (the kmer). and then identifying the pseudo palindromic site surrounding the kmer site. This site can then be resolved into the predicted RRS-1 and RRS-2 motifs.
  • the LSR is LSR 004, (LSR protein ID 2955954; SEQ ID NO: 16) and the kmer is SEQ ID NO: 9820
  • the LSR is LSR_005, (LSR protein ID 3530952; SEQ ID NO: 20) and the kmer is SEQ ID NO: 9821.
  • the LSR is LSR 007, (LSR protein ID 3564078; SEQ ID NO: 3) and the kmer is SEQ ID NO: 9822.
  • the LSR is LSR_012, (LSR protein ID 3577396; SEQ ID NO: 18) and the kmer is SEQ ID NO: 9823.
  • the LSR is LSR 020, (LSR protein ID 3132173; SEQ ID NO: 7) and the kmer is SEQ ID NO: 9824.
  • the LSR is LSR 037, (LSR protein ID 2955313, SEQ ID NO: 2050) and the kmer is SEQ ID NO: 9825.
  • the LSR is LSR_057, (LSR protein ID 3249940: SEQ ID NO: 1319) and the kmer is SEQ ID NO: 9826.
  • the LSR is LSR 078, (LSR protein ID 3281695: SEQ ID NO: 1944) and the kmer is SEQ ID NO: 9827 Tn
  • the LSR is LSR_086, (LSR protein ID 3580517; SEQ ID NO:
  • the LSR is LSR_I50, (LSR protein ID 3537784; SEQ ID NO: 591) and the kmer is SEQ ID NO: 9836.
  • the LSR is LSR 175, (LSR protein ID 3531245; SEQ ID NO: 350) and the kmer is SEQ ID NO: 9837
  • the LSR is LSR_232/LSR_509, (LSR protein ID 3507550; SEQ ID NO: 441) and the kmer is SEQ ID NO: 9838.
  • the LSR is LSR_253, (LSR protein ID 3568126; SEQ ID NO: 620) and the kmer is SEQ ID NO: 9839.
  • the LSR is LSR_263, (LSR protein ID 3474531 ; SEQ ID NO: 1 105) and the kmer is SEQ ID NO: 9840
  • the LSR is LSR_300/LSR_699, (LSR protein ID 3459461 ; SEQ ID NO: 2068) and the kmer is SEQ ID NO: 9841.
  • the LSR is LSR .625, (LSR protein ID 3139148: SEQ ID NO: 1735) and the kmer is SEQ ID NO: 9842.
  • the LSR is LSR 662, (LSR protein ID 3445527; SEQ ID NO: 1162) and the kmer is SEQ ID NO: 9843.
  • the present disclosure systems for precision editing and uses thereof.
  • components of the system are provided in a single composition.
  • Such systems may be referred to as precision editing systems.
  • Precision editing systems may be useful for generating an RRS in a target nucleic acid.
  • a target nucleic acid may not initially contain an RRS that can be utilized by an LSR system.
  • a precision editing sy stem described herein could be used to generate an RRS in the target nucleic acid, thereby making it possible to insert a donor nucleic acid at that site with an LSR.
  • Systems, compositions, and methods disclosed herein may comprise an LSR or nucleic acid encoding the LSR, and a precision editing system; and uses thereof.
  • precision editing systems comprise an RNA-dependent DNA polymerase (RDDP), an effector protein (e.g,, a CRISPR associated (Cas) protein), a guide RNA, and a template RNA.
  • RDDP RNA-dependent DNA polymerase
  • Cas CRISPR associated
  • the RDDP and the effector protein are covalently linked.
  • the guide RNA and template RNA (retRNA) are fused or linked as an extended guide RNA rtgRNA).
  • the present disclosure provides methods of modifying target nucleic acids utilizing precision editing systems. See FIGs. 3-6 for non-limiting depictions of exemplary precision editing systems.
  • precision editing systems comprise: (a) an effector protein or a nucleic acid encoding the effector protein: (b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; (c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises (i) a first region comprising a protein binding sequence, and (ii) a second region comprising a spacer sequence that hybridizes to a target sequence of a first strand of a double stranded DNA (dsDNA) target nucleic acid, wherein the first region is located 5’ of the second region; and (d) a template RN A (retRN A) or nucleic acid encoding the retRNA, wherein the retRNA comprises (i) a primer binding sequence (PBS), and (ii) a template sequence that hybridizes to the target sequence of a second strand of the dsDNA target
  • the guide RNA is linked to the retRNA.
  • the template sequence is located 5’ of the PBS, optionally wherein the 3’ end of the PBS is linked to the 5’ end of the template sequence.
  • the retRNA is circularized.
  • the template sequence comprises an RRS sequence or a sequence that is reverse complementary to an RRS.
  • the RRS may be an RRS that is recognized by an LSR described herein.
  • the one or more RRSs is linked to a primer binding sequence.
  • the guide nucleic acid may interact with an effector protein and target the effector protein to the desired location in the cell genome.
  • the effector protein may nick a strand of the cell genome and the RDDP may incorporate the one or more RRSs of the guide nucleic acid into the nicked site. This provides an RRS site at the desired location of the cell genome.
  • the RDDP is not fused to the effector protein.
  • the RDDP is fused to an aptamer binding protein
  • the guide RNA and/or retRNA comprises an aptamer that is capable of being bound by the aptamer binding protein.
  • a non-limiting example of an aptamer is an MS2 aptamer and a non-limiting example of a corresponding aptamer binding protein is an MS2 coat protein. Additional examples of such localizing systems are described by Chen et al., FEBS J. (2013) 280:3734-3754.
  • the fusion protein described herein comprises an effector protein comprising an ammo acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an ammo acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9402-9482
  • the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267- 9401 and an RDDP comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9402-9482.
  • the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9413.
  • the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9413.
  • the fusion protein comprises an ammo acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9702 or 9707.
  • the fusion protein comprises or consists of SEQ ID NO: 9702 or 9707.
  • the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9415.
  • the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9415.
  • the fusion protein comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9703 or 9708.
  • the fusion protein comprises or consists of SEQ ID NO: 9703 or 9708.
  • the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9421.
  • the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9423.
  • the fusion protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9705 or 9710. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 9705 or 9710.
  • the fusion protein described herein comprises an effector protein comprising an ammo acid sequence that is at least 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9431.
  • the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9267-9401 and an RDDP comprising or consisting of the amino acid sequence of SEQ ID NO: 9431.
  • the fusion protein comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9706 or 9711.
  • the fusion protein comprises or consists of SEQ ID NO: 9706 or 9711.
  • the biological tether or protein localization sequence is MS2, Csy4 or lambda N protein.
  • the effector proteins are complexed with a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2. coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the Type V effector protein guide RNA targeting sequences.
  • a Type V effector protein variant fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (IncRNA) such as XIST or HOTAIR; see. e g., Keryer-Bibens et al.. Biol. Cell 100: 125- 138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence.
  • the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the protein can be targeted to the dCpfl variant binding site using the methods and compositions described herein.
  • the Csy4 is catalytically inactive.
  • the RDDP comprises a reverse transcriptase.
  • the RDDP comprises an amino acid sequence that is at least 75%, at least 80%, al least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.
  • the effector protein nicks a strand of the target nucleic acid and the RDDP synthesizes new DMA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid.
  • the effector protein nicks the target strand.
  • the effector protein nicks the non-target strand.
  • the effector protein is a Cas protein.
  • the effector protein is a Type V effector protein
  • the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 9267-9401 and 9698-9701 . Additional effector proteins described herein and throughout are useful in precision editing systems.
  • the effector protein nicks both the target strand and the nontarget strand sequentially.
  • the effector protein may nick the target strand first and facilitate, with an rtgRNA or retRNA, the introduction of a desired genomic edit.
  • the effector protein, m combination with a guide RNA that targets the non-target strand can nick the non-target strand.
  • Tire cellular DNA repair mechanisms can then repair the non-target strand using the edited target strand as template.
  • systems comprise (a) an I, SR comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%. at least 99%, or 100% identical to SEQ ID NOs: 1-3918 and 9761-9763; (b) an effector protein comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%.
  • an RDDP comprising an ammo acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%>, at least 99%, or 100% identical to SEQ ID NOs: 9402-9482.
  • the systems provided herein comprise (a) a fusion protein described herein (or a polynucleotide encoding the same); and (b) an rtgRNA comprising a guide RNA and a retRNA.
  • the systems provided herein comprise an RDDP comprising (a) an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 9402-9482; (b) an effector protein; and (c) an rtgRNA comprising a guide RNA and a retRNA.
  • the systems provided herein comprise (a) a fusion protein described herein (or a polynucleotide encoding the same); (b) an rtgRNA comprising a guide RNA and a template RNA; and (c) a guide RNA.
  • the systems provided herein comprise an RDDP comprising (a) an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 9402-9482: (b) an effector protein; (c) an rtgRNA comprising a guide RNA and a retRNA; and (d) a guide RNA.
  • the enzyme that is capable of catalyzing the modification of the target nucleic acid forms a complex with an extended guide RNA (rtgRNA).
  • the extended guide RNA comprises (not necessarily in this order): a first region (also referred to as a protein binding region, protein binding sequence, or a repeat sequence) that interacts with an effector protein; a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; a third region comprising a template sequence that is complementary to at least a portion of the target sequence on the non-target strand of the target dsDNA molecule with the exception of at least one nucleotide: and a fourth region comprising a primer binding sequence that hybridizes to a primer sequence of the target dsDNA molecule that is formed when target nucleic acid is cleaved.
  • the third region or template sequence may comprise a nucleotide having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when the template sequence and the target sequence are aligned for maximum identity.
  • the linker comprises a nucleotide. In some embodiments, the linker comprises multiple nucleotides.
  • the third and fourth regions are 5’ of the first and second regions.
  • the order of the regions of the extended guide RNA from 5’ to 3‘ is: third region, fourth region, first region, and second region
  • the effector protein is fused to an RDDP.
  • the RDDP comprises a reverse transcriptase. See, e.g., FIG. 3.
  • the effector protein may nick a strand of the target nucleic acid and the RDDP synthesizes new DNA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid.
  • the effector protein nicks the target strand.
  • the effector protein nicks the non-target strand.
  • the third and fourth regions are 3’ of the first and second regions.
  • the order of the regions of the extended guide RNA from 5’ to 3' is: first region, second region, third region, and fourth region. In some embodiments, there is a linker between the second and third regions.
  • the effector protein is fused to an RDDP.
  • the RDDP comprises a reverse transcriptase. See, e.g. ,
  • compositions and systems comprise (1) a guide RNA comprising (a) a first region (also referred to as a protein binding region, protein binding sequence, or repeat sequence) that interacts with an effector protein and (b) a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; and (2) a template RNA (retRNA) comprising (a) a.
  • a guide RNA comprising (a) a first region (also referred to as a protein binding region, protein binding sequence, or repeat sequence) that interacts with an effector protein and (b) a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; and (2) a template RNA (retRNA) comprising (a) a.
  • a guide RNA comprising (a) a first region (also referred to as a protein binding region, protein binding sequence, or repeat sequence) that interacts with an effect
  • primer binding sequence that hybridizes to a primer sequence of the target dsDNA molecule that is formed when the target nucleic acid is cleaved and (b) a template sequence that is complementary to at least a portion of the target sequence on the second strand of the target dsDNA molecule with the exception of at least one nucleotide.
  • the template sequence may comprise a nucleoli de having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when the template sequence and the target sequence are aligned for maximum identity
  • the primer binding sequence is linked to the template sequence.
  • the guide RNA and the template RNA are covalently connected see, e.g. FIGs. 3-4.
  • the guide RNA and the template RNA are not covalently connected, see, e.g. FIGs, 5A-5B and FIG. 6.
  • such compositions comprise an effector protein that is fused to a RDDP
  • such compositions comprise an effector protein that is not linked or fused (e.g . covalently linked) to an RDDP.
  • Compositions and systems that comprise an RDDP that is not Imked/fused to an effector protein and/or a guide RNA that is not fused/linked to a template RNA may be referred to as a "‘split protein/RNA design" See, e.g. FIGs. 5A-5B and FIG.
  • the effector protein may nick a strand of the target nucleic acid and the RDDP synthesizes new DNA off the nicked end, wherein the new DNA is complementary to the template sequence (RT template), thereby producing the desired genomic edit in the target nucleic acid.
  • the effector protein nicks the target strand In some embodiments, the effector protein nicks the non-target strand.
  • the present disclosure systems for base editors and uses thereof.
  • Base editors may be useful for generating an RRS in a target nucleic acid.
  • a target nucleic acid may not initially contain an RRS that can be utilized by an LSR system
  • a base editor described herein could be used to generate an RRS in the target nucleic acid, thereby making it possible to insert a donor nucleic acid at that site with an LSR.
  • Systems, compositions, and methods disclosed herein may comprise an LSR or nucleic acid encoding the LSR, and a base editor; and uses thereof.
  • fusion partners edit anucleobase of a target nucleic acid. Fusion proteins comprising such a fusion partner and an effector protein may be referred to as base editors. Such a fusion partner may be referred to as a base editing enzyme.
  • a base editor comprises a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme van ant.
  • abase editor may be a fusion protein comprising a base editing enzyme linked to an effector protein.
  • the amino terminus of the fusion partner protein is linked to the carboxy terminus of the effector protein by the linker.
  • the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker.
  • the base editor may be functional when the effector protein is coupled to a guide nucleic acid.
  • the guide nucleic acid imparts sequence specific activity to the base editor.
  • the effector protein may comprise a catalytically inactive effector protein (e g., a catalytically inactive variant of an effector protein described herein),
  • the base editing enzyme may comprise deaminase activity. Additional base editing enzymes are described herein.
  • base editing enzymes are capable of catalyzing editing (e g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DMA or RNA (single stranded or double stranded).
  • a base editing enzyme, and therefore a base editor comprising the same is capable of converting an existing nucleobase to a different nucleobase.
  • base editors edit a nucleobase on a ssDNA. In some embodiments, base editors edit a nucleobase on both strands of dsDNA. In some embodiments, base editors edit a nucleobase of an RNA.
  • a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase
  • a base editing enzyme upon binding to its target locus in the target nucleic acid (e.g., a DNA molecule), base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop’ r .
  • DNA bases within the R-loop are edited by the base editor having the deaminase enzyme activity.
  • base editors for improved efficiency in eukary otic cells comprise a catalytically inactive effector protein that may' generate a nick in the non-edited strand, inducing repair of the non-edited strand using the edited strand as a template.
  • a base editor comprises a deaminase enzyme.
  • Exemplary' deaminases are described in US20210198330. WO2021041945, WO2021050571 Al, and W02020123887, all of which are incorporated herein by reference in their entirety'.
  • Exemplary deaminase domains are described WO 2018027078 and W02017070632, and each are hereby incorporated in its entirety by reference.
  • additional exemplary deaminase domains are described in Komor et al.. Nature, 533, 420-424 (2016): Gaudelli et al.. Nature, 551.
  • the deaminase is ADAR1/2, ADAR-2, AID, or any functional variant thereof
  • the deaminase functions as a monomer.
  • the deaminase functions as heterodimer with an additional protein.
  • base editors comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)).
  • UMI uracil glycosylase inhibitor
  • UNG uracil N-glycosylase
  • a base editor is a cytosine base editor (CBE) and comprises a cytosine base editing enzyme.
  • the cytosine base editing enzyme may convert a cytosine to a thy mine.
  • a cytosine base editing enzyme may accept ssDNA as a substrate but may not be capable of cleaving dsDNA, as linked to a catalytically inactive effector protein.
  • the catalytically inactive effector protein of the CBE when bound to its cognate DNA, may perform local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single-stranded bubble
  • the catalytically inactive effector protein generated ssDNA R-loop may enable the CBE to perform efficient and localized cytosine deamination in vitro.
  • deamination activity is exhibited in a window of about 4 to about 10 base pairs.
  • fusion to the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which may enable the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies.
  • the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo.
  • the cytosine base editing enzyme is a cytidine deaminase.
  • the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2016) Nature Biotechnology' 36:848-846; Komor et al (2016) Nature 533:42.0-
  • CBEs comprise a uracil glycosylase inhibitor (UGI) or uracil N- glycosylase (UNG).
  • base excision repair (BER) of U»G in DNA is initiated by a UNG, which recognizes a U»G mismatch and cleaves the gly osidic bond between a uracil and a deoxyribose backbone of DNA.
  • BER results in the reversion of the U»G intermediate created by the first CBE back to a C»G base pair.
  • the UNG may be inhibited by fusion of a UGI.
  • the CBE comprises a UGI.
  • a C -terminus of the CBE comprises the UGI.
  • the UGI is a small protein from bacteriophage PBS.
  • the UGI is a DNA mimic that potently inhibits both human and bacterial UNG
  • the UGI inhibitor is any protein or polypeptide that inhibits UNG.
  • the CBE may mediate efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C «G base pair to a T» A base pair through a U s G intermediate.
  • the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.
  • a CBE nicks a non-edited DNA strand.
  • the non-edited DNA strand nicked by the CBE biases cellular repair of a U «G mismatch to favor a U*A outcome, elevating base editing efficiency.
  • a APOBEC1- nickase-UGI fusion efficiently edits in mammalian cells, while minimizing frequency of nontarget indels.
  • base editors do not comprise a functional fragment of the base editing enzyme. Tn some embodiments, base editors do not comprise a functional fragment of a UGI, where such a fragment may be capable of excising a uracil residue from DNA by cleaving an N-glycosidic bond.
  • the base editor further comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI).
  • npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG.
  • the npUGI is a small molecule deri ved from uracil. Examples of small molecule non-protein uracil-DNA glycosylase inhibitors, fusion proteins, and Cas- CR1SPR systems comprising base editing activity are described in WO2021087246. which is incorporated by reference in its entirety.
  • the cytosine base editing enzyme is a cytidine deaminase.
  • the cytidine deaminase base editing enzy me is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety.
  • Non-limiting exemplary' cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D,
  • BE3 APOBEC !-XTEN-dCas9(A840H)-UGI
  • WO2021087246, WO2021062227, and WO2020123887 which are incorporated herein by reference in their entirety.
  • a base editor is a cytosine to guanine base editor (CGBE) and comprises a cytosine to guanine base editing enzyme.
  • CGBE may convert a cytosine to a guanine.
  • a base editor is an adenine base editor (ABE) and comprises an adenine base editing enzyme.
  • An ABE may convert an adenine to a guanine.
  • an ABE converts an A*T base pair to a G*C base pair.
  • the ABE converts a target A»T base pair to G»C in vivo or in vitro.
  • ABEs provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations.
  • ABEs provided herein enable correction of pathogenic SNPs (-47% of disease-associated point mutations).
  • the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine In some embodiments, inosine exhibits the base-pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation.
  • Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, AP0BEC3A. Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2.
  • Non- limiting exemplary' ABEs suitable for use herein include: ABE7. ABE8.1m, ABES.2m.
  • the base editing enzyme alters one base of the nucleotide sequence io make the nucleotide sequence into a recombinase recognition sequence
  • the base editing enzy me alters two or more bases of the nucleotide sequence to make the nucleotide sequence into a recombinase recognition sequence.
  • multiple base editing enzymes are used to alter a nucleotide sequence that is not a recombinase recognition sequence into a nucleotide sequence that is a recombinase recognition sequence.
  • compositions, systems, raid methods comprising an effector protein and uses thereof
  • the effector protein comprises a CRISPR associated (Cas) protein.
  • the Cas protein comprises a Type II Cas protein.
  • the Cas protein does not comprise a Type II Cas protein.
  • the effector protein does not comprise a Cas9 protein
  • the Cas protein comprises a Type V Cas protein
  • the Type V Cas protein comprises an RuvC domain and does not comprise an HNH domain.
  • the Cas protein comprises a Type VU-3 Cas protein.
  • the Cas protein comprises a Type VU-4 protein.
  • the Cas protein comprises a CasPhi (Cas®, Casl2J) protein In some embodiments, the Cas protein comprises a Cas 14 protein. In some embodiments, the Casl4 protein is a Casl4a protein. In some embodiments, the Casl4 protein is a Casl4b protein. In some embodiments, the Cas protein is CasM.265466.
  • effector proteins described herein comprise one or more functional domains.
  • Effector protein functional domains may include a protospacer adjacent motif (PAM)- interacting domain, an oiigonucleotide-mteracting domain, one or more recognition domains, a non-target strand interacting domain, and a RuvC domain.
  • a PAM interacting domain can be a target strand PAM interacting domain (TPID) or anon-target strand PAM interacting domain (NTPID).
  • TPID target strand PAM interacting domain
  • NTPID non-target strand PAM interacting domain
  • a PAM interacting domain, such as a TPID or a NTPID, on an effector protein descri bes a region of an effector protein that interacts with target nucleic acid.
  • the effector proteins comprise a RuvC domain.
  • a RuvC domain comprises with substrate binding activity, catalytic activity, or both.
  • the RuvC domain may be defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein.
  • An effector protein of the present disclosure may include multiple RuvC subdomains, which may combine to generate a RuvC domain with substrate binding or catalytic activity.
  • an effector protein may include three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary' amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds.
  • effector proteins comprise one or more recognition domain (REC domain) with a. binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex.
  • An effector protein may comprise a zinc finger domain.
  • the effector protein does not comprise an HNH domain.
  • an effector protein is similar to a naturally occurring effector protein
  • the effector protein may lack a portion of the naturally occurring effector protein.
  • the effector protein may comprise a mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature.
  • the effector protein may also comprise at least one additional amino add relative to the naturally-occurring effector protein.
  • the effector protein may comprise an addition of a nuclear localization signal relative to the natural occurring effector protein.
  • a nucleotide sequence encoding the effector protein is codon optimized ( ⁇ ?.g., for expression in a eukaryotic cell) relative io the naturally occurring sequence.
  • the length of the effector protein is about 350 to about 450 linked amino acid residues In some embodiments, the length of the effector protein is about 375 to about 475 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 450 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 500 linked amino acid residues.
  • the length of the effector protein is about 350 to about 400, about 400 to about 450, about 450 to about 550, about 400 to about 420, about 420 to about 440, about 440 to about 460, about 460 to about 480, about 480 to about 500, about 500 to about 520, about 520 to about 540, about 540 to about 560, about 560 to about 580, about 580 to about 600, about 600 to about
  • compositions, systems, and methods provided herein comprise an effector protein and an engineered guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to any one of the sequences as set forth in TABLE 4.
  • a mutation may affect the catalytic activity of the effector protein and results in a catalytically reduced or catalytically inactive mutant. In some embodiments, a mutation can result in the effector protein having nickase activity or increased nickase activity. In some embodiments, a mutation can result in the effector protein having reduced or no nuclease activity' but gaming nickase activity.
  • CasPhi CasPhi
  • the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9694.
  • the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, ai least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. or 100% identical to the sequence:
  • the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or
  • the effector protein is CasPhi. 12 (SEQ ID NO: 9278) or a variant thereof described herein
  • systems and compositions comprise an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9278, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.
  • Effector proteins of the present disclosure, dimers thereof, and niultimeric complexes thereof, may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • PAM protospacer adjacent motif
  • cleavage occurs within 1, 2, 3. 4. 5, 6. 7, 8, 9. 10, I L 12. 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, or 25 nucleotides of a 5’ or 3’ terminus of a PAM sequence
  • a target nucleic acid may comprise a PAM sequence adjacent to a target sequence that is complementary to a guide nucleic acid spacer region.
  • PAMs in compositions, systems, and methods herein are further described throughout the application.
  • the PAM is located 5’ of the target sequence on the non-target strand of the target nucleic acid.
  • the PAM is located 3’ of the target sequence on the target strand of the target nucleic acid
  • a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence, wherein the PAM sequence comprises any one of the PAM sequences as set forth in TABLE 4.
  • systems, compositions, and/or methods described herein comprise a target nucleic acid comprising a target sequence that is adjacent to a PAM sequence, wherein the PAM sequence comprises any one of the PAM sequences as set forth in TABLE 4.
  • RNA-dependent DNA polymerases RDDP
  • the present disclosure provides for systems, compositions, and methods comprising an RNA-dependent DNA polymerase (RDDP) or a use thereof.
  • RDDPs are enzymes that are capable of generating a DNA polynucleotide from an RNA template polynucleotide
  • the RDDP is a reverse transcriptase.
  • a nonlimiting example of a reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase (e.g., SEQ ID NO: 9471 or 9472).
  • M-MLV Moloney murine leukemia virus
  • the term RDDP encompasses functional domains thereof.
  • the functional domain is a polymerase domain, an RNAse domain, or a combination thereof.
  • the RDDP comprises less than 800 amino acids. In some embodiments, the RDDP comprises less than 800, less than 700, less than 600, less than 500, less than 400. less than 300 ammo acids. In some embodiments, the RDDP comprises at least 200, at least 220, at least 2.40, at least 260, at least 280, or at least 300 amino acids. In some embodiments, the RDDP comprises at least 277 amino acids. In some embodiments, the RDDP comprises 200 to 800 amino acids. In some embodiments, the RDDP comprises 277 to 800 amino acids. In some embodiments, the RDDP comprises 300 to 800. 400 to 800, 500 to 800, or 600 to 800 amino acids. In some embodiments, the RDDP comprises 250 to 750, 250 to 700, 250 to 650. 250 to 600, 250 to 550. 250 to 500, 250 to 450, 250 to 400, 250 to 350, 275 to 750,
  • the length of the RDDP is less than 800 amino acids. In some embodiments, the length of the RDDP is less than 800, less than 700, less than 600, less than
  • the length of the RDDP is at least 200, at least 220. at least 240, at least 260. at least 280, or at least 300 linked ammo acids. In some embodiments, the length of the RDDP is at least 277 linked amino acids. In some embodiments, the length of the RDDP is 200 to 800 linked ammo acids. In some embodiments, the length of the RDDP is 277 to 800 linked amino acids. In some embodiments, the length of the RDDP is 300 to 800, 400 to 800, 500 to 800, or 600 to 800 linked amino acids. In some embodiments, the length of the RDDP is 250 to 750.
  • the polynucleotide encodes an RDDP comprising or consisting of an amino sequence selected from SEQ ID NOs: 9402-9471.
  • Exemplary RDDP sequences are provided in TABLE 8 below.
  • RDDPs comprise al least 200. at least 225. at least 250. at least 275 at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at. least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725. at least 750, at least 775 contiguous amino acids of a sequence selected from SEQ ID NOs: 9402-9471.
  • effector proteins described herein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal ceil, a mammalian cell, or a human cell.
  • the effector protein is codon optimized for a human cell.
  • LSRs, RDDPs, and/or effector proteins described herein comprise one or more amino acid substitutions as compared to a naturally occurring protein.
  • the amino acid substitution is a conservative amino acid substitution.
  • a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).
  • Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (group 1 to 6) for another amino acid of the same group.
  • Amino acid residues may be divided into groups based on common side chain properties, as follows: (group 1) hydrophobic: norieucine (Nle), methionine (Met), Alanine (Ala), Valine (Vai), Leucine (Leu). Isoleucine (lie); (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr). Asparagine (Asn), Glutamine (Gin); (group 3) acidic: Aspartic acid (Asp). Glutamic acid (Glu); (group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg); (group 5) residues that influence chain orientation: Glycine (Gly).
  • the substitution of one amino acid with another amino acid in a same group listed above may be considered a conservative amino acid substitution.
  • the substitution of one amino acid with another amino acid in a different group listed above may be considered a non-conservative ammo acid substitution.
  • the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is at aposition selected from K58, 180, T84, KI 05, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof.
  • the poly peptide comprises an amino acid sequence that is 1 ( )( )% identical to SEQ ID NO: 9401, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the ammo acid substitution is a position selected from K58, 180, 1'84, KI 05, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof.
  • the amino acid substitution is selected from K58X, 180X, T84X, K105X. N193X. C202X, S209X. G210X. A218X, D220X, E225X, C246X. N286X, M295X, M298X. A306X, Y315X. and Q360X. wherein X is selected from
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%. at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R. A218R, D220R, E225R, C246R, Q360R, I80K, T84K. G210K, N193K, C202K.
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9401. with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from T80R.
  • these engineered effector proteins demonstrate enhanced nuclease activity' relative to the wild-type effector protein.
  • the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9401, wherein the polypeptide comprises at least one ammo acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from D237A, D418A, D418N. E335A, and E335Q, and a combination thereof
  • the polypeptide comprises an amino add sequence that is 100% identical to SEQ ID NO: 9401, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9401, wherein the amino acid substitution is selected from D237A.
  • catalytically inactive effector protein also referred to as a “‘dCas” protein, as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity 7 relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid.
  • the catalytic activity that is reduced or eliminated is often a nuclease activity.
  • the naturally-occurring effector protein may be a wildtype protein.
  • the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.
  • the catalytically inactive effector protein is referred to as a dead Cas protein or a dCas protein ,
  • the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%. or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is at a position selected from 12, T5, KI 5, Rl 8, H20, S21, L26, N30, E33, E34. A35, K37, K38, R41, N43, Q54, Q79R, K92E. K99R, S 108, E1 Q9, HHO. Gi l l , D I 13, Tl 14, P H6. K i i8. El 19, A12I , N 132. K135, Q138, V I 39, N148, LI 49, E157, El 64, El 66, El 70, Y180, LI 82,
  • the polypeptide comprises an ammo acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is at a position selected from 12, T5, KI 5, Rl 8. H20, S21 , L26, N30, E33, E34, A35, K37. K38. R41,
  • the amino acid substitution is selected from I2X, T5X. K15X, R18X, H20X, S21X, L26X. N30X, E33X, E34X, A35.X, K37X. K38X. R41X, N43X, Q54X, Q79RX, K92EX, K99RX, S108X, E109X, H110X, Gi l IX, D113X, T1 I4X.
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, 147 IT, S579R, F701R. P707R, K189P, S638K, Q54R, Q79R, Y220S. N406K, El 19S, K92E.
  • the polypeptide comprises an amino acid sequence tha t is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, 147 IT, S579R, F701R.
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9278.
  • polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from L26K/A121Q, L26R/A121Q, K99R/L149R, K99R/N148R, L 149R/H208R, S362R/L26R L26R/N148R, L26R/H208R. N30R/N 148R, L26R/K99R, L26R/P707R,
  • L26R/K435Q L26R/G685R, L26R/Q674K, L26R/P699R, L26R/T70E, L26R/Q232R, L26R/T252R, L26R/P679R, L26R/E83K, L26R/E73P, L26R/K248E, L26R, T5R7 S223P, S579R/ S223P, L26R/ S223P, T5R/ Al 21 Q, L26R/ A696R, S198R/ 147 IT, L26R/ N153R, L26R/ E682R, L26R/ D703R, Q612R' L26K, L26R/ 147 IT, K348R/ L26K, S579R/I471T, L26R/V228R, T5R/S638K, S579R/K189P, S579R/E258K, L26RZ
  • F701R/A121Q L26R/G361R, S198R/E258K, L26R/S472R, T5R/Y220S, L26R/A150K, L26R/S684R, L26R/E157R, L26R/K248R, F701R/L26K, S198R/N406K, S198R/Y220S, SI98R/S638K, S198R/V521T, S579R/A121 Q, K.348R/Y220S.
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one amino acid substitution relative to SEQ
  • amino acid substitution is selected from L26K/A121Q, L26R'A12IQ, K99R/L149R, K99R/N148R. L149R/H208R. S362R''L26R L26R/N148R, L26R/H208R, N30R/N148R, L26R/K99R, L26R/ P7O7R. L26R'L149R. L26R/N30R.
  • L26R/K435Q L26R/G685R, L26R/Q674K, L26R/P699R, L26R/T70E, L26R/Q232R,
  • SI 98R/ 147 IT L26R/ NI53R, L26R/ E682R, L26R/ D703R, Q612R/ L26K, L26R/ 147 IT, K348R/ L26K, S579R/I471T, L26R/V228R, T5R/S638K, S579R/K189P, S579R/E258K, L26R/K260R, L26R/S638K,
  • L26R/C405R S579R/V521T, S579R/N406K. T5R/K92E, T5R/E258K, L26R/I97R, S579R/S638K, T5R/K435Q, F701R/S638K, L26R/L236R, F701R/I471T, Q612R/S223P, F701R/S223P, S198R/E119S, S579R/K92E, L26R/E715R, Q612R/I471T, F70IR/Y220S, S 198R/S223P, and L26R/K266R, and a combination thereof.
  • these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9278.
  • the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 9278 wherein the amino acid substitution is selected from E157A, El 64 A, E164L. E166A, El 661. E170A, I489A, I489S. Y490S, Y490A, F491 A. F491S, F491G, D495G, D495R, D495K, K496A. K496S, K498A.
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9278. with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from EI 57A, E164A, E164L, E166A. El 661, E170A, I489A, I489S, Y490S. Y490A. F491A, F491S, F49IG. D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S,
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9278, wherein amino acids S478-S505 have been deleted, in some embodiments, the effector protein is an engineered effector protein that is at least 90%, at least 95%, at least 97%. at least 98%.
  • the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%. at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 9700. In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 9701.
  • the effector protein is an engineered effector protein and comprises an ammo acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9278, wherein the polypeptide comprises at least one ammo acid substitution relative to SEQ ID NO: 9278 wherein the amino acid substitution is selected from D369A, D369N, D658A, D658N, E567A, E567Q, and a combination thereof.
  • the polypeptide comprises an ammo acid sequence that is 100% identical to SEQ ID NO: 9278, with the exception of at least one ammo acid substitution relative to SEQ ID NO: 9278, wherein the amino acid substitution is selected from D369A, D369N. D658A, D658N, E567A, E567Q, and a combination thereof.
  • these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein TABLE 6 provides the exemplary amino acid alterations relative to SEQ ID NO: 9278 useful in compositions, systems, and methods described herein.
  • the RDDP is an engineered RDDP and comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 9472-9482.
  • the RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 9472-9482.
  • the present disclosure provides a polynucleotide encoding an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 9472-9482.
  • the RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 9472-9482.
  • the present disclosure provides a polynucleotide encoding an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to
  • the present disclosure provides a polynucleotide encoding an RDDP comprising an amino acid sequence that it at least 90%, 91 %. 92%. 93%, 94%, 95%. 96%. 97%, 98%, or 99% identical to one of SEQ ID NOs: 9472- 9482.
  • the polynucleotide encodes an RDDP comprising or consisting of an amino sequence selected from SEQ ID NOs: 9472-9482,
  • RDDPs comprise at least 200, at least 225, at least 250, at least 275 at least 300, at least 325, at least 350, at least 375. at least 400, at least 425. at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775 contiguous amino acids of a sequence selected from SEQ ID NOs: 9472-9482,
  • Exemplary' engineered RDDPs are provided in TABLE 9 below.
  • the parental RDDP is listed followed by the ammo acid mutations in parentheses.
  • 2691319 (D12R-D72R-N195R) refers to an engineered RDDP based on 2691319 (SEQ ID NO: 9413) and comprising the mutations D12R, D72R, and N195R.
  • Engineered effector proteins may provide enhanced catalytic activity (e.g., nuclease or nickase activity) as compared to a naturally occurring nuclease or nickase.
  • Engineered effector proteins may provide enhance nucleic acid binding activity, e.g, enhanced binding of a guide nucleic acid and/or target nucleic acid, and/or may demonstrate a stronger affinity for a target nucleic acid sequence.
  • substitution of positively charged amino acids is thought to increase the interaction between the effector proteins and/or RDDPs and the negatively charged target nucleic acid sequences.
  • some engineered proteins exhibit optimal activity at lower salinity and viscosity than the protoplasm of their bacterial cell of origin.
  • bacteria often comprise protoplasmic salt concentrations greater than 250 mM and room temperature intracellular viscosities above 2 centipoise
  • engineered proteins exhibit optimal activity (e.g., cis-cleavage activity) at salt concentrations below 150 rnM and viscosities below 1.5 centipoise.
  • the present disclosure leverages these dependencies by providing engineered proteins in solutions optimized for their activity and stability.
  • compositions and systems described herein may comprise an engineered effector protein and/or RDDP in a sol ution comprising a room temperature viscosity of less than about 15 centipoise, less than about 12. centipoise, less than about 10 centipoise, less than about 8 centipoise, less than about 6 centipoise, less than about 5 centipoise, less than about 4 centipoise, less than about 3 centipoise, less than about 2 centipoise, or less than about 1.5 centipoise.
  • compositions and systems may comprise an engineered effector protein anchor RDDP in a solution comprising an ionic strength of less than about 500 mM. less than about 400 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 150 mM, less than about 100 mM, less than about 80 mM, less than about 60 mM, or less than about 50 mM.
  • Compositions and systems may comprise an engineered effector protein and/or RDDP and an assay excipient, which may stabilize a reagent or product prevent aggregation or precipitation, or enhance or stabilize adetectable signal (e g., a fluorescent signal).
  • assay excipients include, but are not limited to, saccharides and saccharide derivatives (e.g., sodium carboxy methyl cellulose and cellulose acetate), detergents, glycols, polyols, esters, buffering agents, alginic acid, and organic solvents (e.g , DMSO).
  • An engineered protein may comprise a modified form of a wild type counterpart protein.
  • the modified form of the wild type counterpart may comprise an ammo acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart.
  • an ammo acid change e.g., deletion, insertion, or substitution
  • a nuclease domain e.g., a nuclease domain
  • RuvC domain of an effector protein may be mutated relative to a wild type counterpart effector protein so that it is comprises reduced nuclease activity.
  • the modified form of the effector protein may comprise increased nickase activity relative to that of a wild-type counterpart. Modulation of Effector protein activity
  • the effector proteins of the present disclosure may have nuclease activity or nickase activity' on target nucleic acids. In some embodiments the effector protein of the present disclosure has a combination of the above activities on a target nucleic acid. In some embodiments the cleavage activity of the effector proteins is modulated between nuclease activity 7 and nickase activity- on the target nucleic acid.
  • the cleavage activity of the effector protein on the target nucleic acid is modulated based on the length of the spacer sequence of a guide nucleic acid.
  • the spacer length confers nuclease activity to the effector protein on the target nucleic acid.
  • the spacer length confers nickase activity' to the effector protein on the target nucleic acid.
  • the spacer length is 10-20 nucleotides.
  • the spacer length is 11-17 nucleotides.
  • the spacer length is 14-16 nucleotides.
  • the spacer length is 10, 12, 13, 14, 15. 16 or 17 nucleotides
  • the spacer length is 15 nucleotides. Fusion proteins
  • the present disclosure provides a fusion protein comprising an effector protein, an RDDP. an I SR. or a combination thereof. In some embodiments, the present disclosure provides a fusion protein comprising an effector protein, a base editing enzyme, an LSR, or a combination thereof. In some embodiments, the fusion protein comprises, from N-terminus to C -terminus:
  • an LSR a base editing enzyme, and an effector protein
  • the present disclosure provides a fusion protein comprising an effector protein and an LSR, wherein the effector protein lacks nuclease activity.
  • the effector protein lacks nuclease activity.
  • Exemplary embodiments of effector proteins that lack nuclease activity are provided in TABLE 5 and TABLE 6.
  • the effector protein directs the activity of the LSR to a target nucleic acid sequence in combination with a guide RNA.
  • LSRs. RDDPs, effector proteins and fusion proteins of the present disclosure of the present disclosure may be synthesized, using any suitable method. Additionally, nucleic acids, including mRNA encoding LSRs, RDDPs, effector proteins, and fusion proteins of the present disclosure may be synthesized using suitable methods. LSRs, RDDPs, effector proteins, and fusion proteins of the present disclosure may be produced in vitro or by eukaiyotic cells or by prokaryotic cells. LSRs, RDDPs, effector proteins, and fusion proteins can be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method.
  • LSRs, RDDPs, effector proteins, and fusion proteins can be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method.
  • Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al.. Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer. 2nd ed (2014)).
  • an LSR, RDDP, base editing enzyme, effector protein, and/or fusion protein is an isolated protein.
  • an LSR, RDDP, effector protein, base editing enzyme, and/or fusion protein described herein can be isolated and purified for use in compositions, sy stems. and/or methods described herein. Methods described here can include the step of isolating LSRs, RDDPs.
  • LSR LSR
  • RDDP effector protein
  • base editing enzyme and/or fusion protein provided herein
  • recombinant expression systems precipitation, gel filtration, ionexchange, reverse-phase and affinity chromatography, and the like.
  • Other well-known methods are described in Deutscher et al.. Guide to Protein Purification: Methods in
  • the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al.. Molecular Cloning: A Laboratory' Manual. Third Ed., Cold Spring Harbor Laboratory 7 , New York (2001 ); and Ausubel et al.. Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD ( 1999)).
  • the methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.
  • LSRs. RDDPs, effector proteins, and fusion proteins disclosed herein may be covalently linked or attached to a tag, e g., a purification tag
  • a purification tag, as used herein, can be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which can be its biological source, such as a cell lysate Attachment of the purification tag can be at the N or C terminus of the LSRs, RDDPs, effector proteins, and/or fusion proteins.
  • an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease can be inserted between the purification tag and the LSR, RDDP, effector protein, base editing enzyme, and/or fusion protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag.
  • Purification and/or isolation can be through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Examples of purification tags are as described herein. Guide Nucleic A elds
  • compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof.
  • compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout include DNA molecules, such as expression vectors, that encode a guide nucleic acid.
  • compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid.
  • a guide nucleic acid is a nucleic acid molecule, at least a portion of which may be bound by an effector protein, thereby forming a ribonucleoprotein complex (RNP).
  • Another portion of the guide nucleic acid molecule can comprise a spacer region which is complementary to at least a portion of the target nucleic acid sequence.
  • the guide nucleic acid imparts activity or sequence selectivity to the effector protein.
  • guide nucleic acids can bring the effector protein into proximity of a target nucleic acid.
  • the guide nucleic acid spacer region may hybridize to a target nucleic acid or a portion thereof.
  • a guide nucleic acid and an effector protein form an RNP
  • at least a portion of the RNP binds spacer region, recognizes, and/or hybridizes to a target nucleic acid.
  • a RNP can hybridize, via the spacer region, to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein.
  • a guide nucleic acid may comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more sequence modifications as described herein), and any combinations thereof.
  • a guide nucleic acid may comprise a naturally occurring sequence.
  • a guide nucleic acid may comprise a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, may be different from the sequence of a naturally occurring nucleic acid.
  • the guide nucleic acid may be chemically synthesized or recombinantly produced.
  • Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.
  • Guide nucleic acids while often being referred to as a guide RNA, may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • the guide nucleic acid comprises a hairpin or stem-loop structure that is recognized by the effector protein.
  • the hairpin or stem-loop structure may comprise a repeat region.
  • the guide nucleic acid comprises at least a portion of a tracrRNA sequence.
  • a tracrRNA is a guide nucleic acid that has trans activating activity on a target nucleic acid through a repeat hybridization sequence.
  • guide nucleic acids of the instant di sclosure do not comprise a repeat hybridization sequence.
  • guide nucleic acids of the instant disclosure do not comprise a tracrRNA.
  • the guide nucleic acid does not comprise a tracrRNA sequence but comprises a repeat sequence to which the CasPhi protein binds.
  • the guide nucleic acid comprises a repeat region that interacts with the effector protein
  • the term, “repeat region” may be used interchangeably herein with the term, “repeat sequence.’’
  • an effector protein interacts with a repeat region.
  • an effector protein does not interact with a repeat region.
  • the repeat region is adjacent to the spacer region.
  • the repeat region is followed by the spacer region in the 5’ to 3’ direction. Exemplary’ repeat region sequences for exemplary effector proteins provided herein are shown in TABLE 4.
  • guide nucleic acids comprise a spacer sequence that hybridizes with a target sequence of a target nucleic acid.
  • spacer sequence refers to a region of the guide nucleic acid that hybridizes to a target sequence of a target nucleic acid.
  • spacer sequence and “spacer region” are used interchangeably herein and throughout.
  • Tire spacer sequence may comprise a sequence that is complementary with a target sequence of a target nucleic acid.
  • the spacer sequence is complementary to the target sequence on the target strand of a dsDNA molecule.
  • the spacer sequence is complementary' to the target sequence on the non-target strand of a dsDNA molecule.
  • the spacer sequence can function to direct the guide nucleic acid to the target nucleic acid for detection and/or modification of the target nucleic acid.
  • the spacer sequence may’ be complementary' to a target sequence that is adjacent to a PAM that is recognizable by an effector protein of interest.
  • the spacer sequence is 15-28 linked nucleotides in length.
  • the spacer sequence is 15-26, 15-24. 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length.
  • the spacer sequence is 18-24 linked nucleotides in length.
  • the spacer sequence is at least 15 linked nucleotides in length.
  • the spacer sequence is at least 16, 18, 20, or 22 linked nucleotides in length. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. or 30 nucleotides. In some embodiments, the spacer sequence is at least 17 linked nucleotides in length. In some embodiments, the spacer sequence is at least 18 linked nucleotides in length. In some embodiments, the spacer region is at least 20 linked nucleotides in length. In some embodiments, the spacer sequence is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the target nucleic acid. In some embodiments, the spacer sequence is 100% complementary' to the target sequence of the target nucleic acid. In some embodiments, the spacer sequence comprises at least 15 contiguous nucleotides that are complementary' to the target nucleic acid.
  • sequence of a spacer sequence need not be 100% complementary' to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence.
  • the spacer sequence may comprise at least one nucleotide that is not complementary' to the corresponding nucleotide of the target sequence. In some embodiments the spacer sequence is less than 100% complementary' to the target sequence, but still can bind to the target sequence. In some embodiments the spacer sequence is 90%, 91%, 92%, 93%, 94%. 95%. 96%, 97%, 98%, 99%, or 100% complementary to the target sequence. In some embodiments, the target sequence is selected from SEQ ID NO: 8965-9266.
  • a guide nucleic acid, a spacer region thereof or a spacer sequence thereof comprises at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary'' to a eukaryotic sequence.
  • a eukaryotic sequence is a sequence of nucleotides that is present in a host eukaryotic cell.
  • Such a sequence of nucleotides i s distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses.
  • a guide nucleic acid comprises a spacer length that confers nickase activity to a Cas enzyme that otherwise comprises nuclease activity when used with guide nucleic acids having a different spacer length.
  • the Cas enzyme is a Type V Cas enzyme.
  • the Cas enzyme is a CasPhi enzyme.
  • the Cas enzyme is CasPhi. 12 (SEQ ID NO: 9278).
  • the spacer length that confers nickase activity is 14-16 nucleotides. In some cases, the spacer length is 15 nucleotides.
  • a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers.
  • the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers.
  • the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers.
  • the guide nucleic acid comprises two or more linkers In some embodiments, at least two or more linkers are the same. In some embodiments, at least two or more linkers are not same.
  • a linker comprises one to ten, one to seven, one to five, one to three, two to ten. two to eight, two to six. two to four, three to ten. three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides.
  • the linker comprises one. two, three, four, five, six, seven, eight, nine, or ten linked nucleotides.
  • a linker comprises a nucleotide sequence of 5‘-GAAA-3‘ (SEQ ID NO: 9695).
  • a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker.
  • Guide nucleic acids described herein may comprise one or more repeat sequences.
  • a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid.
  • a repeat sequence comprises a nucleotide sequence that may interact with an effector protein
  • a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex). Tn some embodiments, the repeat sequence may also be referred to as a “protein-binding segment.’’’
  • a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3‘ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and/or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence, which may be a direct link or by any suitable linker, examples of which are described herein.
  • the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same polynucleotide molecule.
  • a spacer sequence is adjacent to a repeat sequence.
  • a spacer sequence follows a repeat sequence in a 5’ to 3’ direction.
  • a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction.
  • the spacer(s) and repeat sequence(s) are linked directly to one another.
  • a linker is present between the spacer(s) and repeat sequence(s). Linkers may be any suitable linker.
  • the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate polynucleotide molecules, which are joined to one another by base pairing interactions.
  • the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex).
  • dsRNA duplex double stranded RNA duplex
  • the two sequences are not directly linked and hybridize to form a stem loop structure.
  • the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp)
  • not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence may include a bulge.
  • the repeat sequence comprises a hairpin or stem-loop structure, optionally at the 5’ portion of the repeat sequence.
  • a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary.
  • such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity).
  • a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. , a bulge, a loop structure or hairpin structure, etc.).
  • Exemplary repeat sequences are provided in TABLE 4.
  • Guide nucleic acids described herein may comprise one or more intermediary sequences.
  • an intermediary 7 sequence used in the present disclosure is not transactivated or transactivating.
  • An intermediary sequence may also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides instead of or in addition to ribonucleotides, and/or modified bases.
  • the intermediary' sequence non-covalently binds to an effector protein.
  • the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.
  • an intermediary RNA is a nucleotide sequence in a handle sequence, wherein the intermediary RNA sequence is capable of, at least partially, being non- covalently bound to an effector protein to form a complex (e.g. , an RNP complex).
  • a complex e.g. , an RNP complex
  • an intermediary RNA sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein.
  • a length of the intermediary' sequence is at least 30. 50, 70, 90, 1 10. 130, 150, 170, 190. or 210 linked nucleotides. In some embodiments, a length of the intermediary sequence is not greater than 30, 50, 70, 90, 1 10, 130, 150, 170. 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180. or about 150 to about 180 linked nucleotides.
  • An intermediary’ sequence may' also comprise or form a secondary structure (e.g , one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region).
  • An intermediary’ sequence may comprise from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region of the intermediary’ sequence does not hybridize to the 3‘ region.
  • the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.
  • an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length Tn some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length.
  • an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure).
  • An effector protein may interact with an intermediary sequence comprising a single stem region or multiple stem regions, in some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, an intermediary sequence comprises 1 , 2, 3. 4, 5 or more stem regions.
  • compositions, systems and methods described herein comprise the nucleic acid, wherein the nucleic acid comprises a handle sequence.
  • the handle sequence comprises an intermediary sequence.
  • the intermediary sequence is at the 3’-end of the handle sequence.
  • the intermediary sequence is at the 5’- end of the handle sequence.
  • the handle sequence further comprises one or more of linkers and repeat sequences.
  • the linker comprises a sequence of 5’-GAAA-3’ (SEQ ID NO: 9695).
  • the intermediary sequence is 5’ to the repeat sequence.
  • the intermediary' sequence is 5’ to the linker.
  • the intermediary sequence is 3’ to the repeat sequence. In some embodiments, the intermediary sequence is 3 ‘ to the linker. In some embodiments, the repeat sequence is 3’ to the linker. In some embodiments, the repeat sequence is 5’ to the linker.
  • a sgRNA may include a handle sequence having a hairpin region, as well as a linker and a repeat sequence.
  • the sgRNA having a handle sequence can have a hairpin region positioned 3’ of the linker and/or repeat sequence.
  • the sgRNA having a handle sequence can have a hairpin region positioned 5’ of the linker and/or repeat sequence.
  • the hairpin region may include a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.
  • an effector protein may recognize a secondary structure of a handle sequence.
  • at least a portion of the handle sequence interacts with an effector protein described herein
  • at least a portion of the intermediary sequence interacts with the effector protein described herein.
  • both, at least a portion of the intermediary sequence and at least a portion of the repeat sequence interacts with the effector protein.
  • the handle sequence is capable of interacting (e.g., non-covalent binding) with any one of the effector proteins described herein.
  • the handle sequence of a sgRNA comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • the sgRNA comprises a pseudoknot (e.g., a secondary' structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure).
  • An effector protein may recognize a sgRNA comprising multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the sgRNA comprises at least 2, at least 3. at least 4, or at least 5 stem regions.
  • a handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a length of the handle sequence is at least 30, 50, 70, 90, 1 10, 130, 150, 170. 190, or 210 linked nucleotides.
  • a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190. or 210 linked nucleotides.
  • the length of the handle sequence is about 30 to about 210. about 60 to about 210, about 90 to about 210.
  • the length of a handle sequence in a sgRNA is not greater than
  • the length of a handle sequence in a sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides.
  • the length of a handle sequence in a sgRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 66 to 105 linked nucleotides, 67 to 105 linked nucleotides, 68 to 105 linked nucleotides, 69 to 105 linked nucleotides, 70 to 105 linked nucleotides, 71 to 105 linked nucleotides, 72 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides.
  • the length of a handle sequence in a sgRNA is 40 to 70 nucleotides.
  • the length of a handle sequence in a sgRNA is 50, 56, 66, 67, 68. 69, 70, 71, 72, 73, 95, or 105 linked nucleotides.
  • compositions, systems, and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins.
  • a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein.
  • a second region (SR) of the grade nucleic acid hybridizes with a target sequence of the target nucleic acid.
  • guide nucleic acid comprises a crRNA comprising a spacer sequence(s) and a repeat sequence(s) present within the same polynucleotide molecule.
  • the spacer sequence is adjacent to the repeat sequence.
  • the spacer sequence follows the repeat sequence in a 5’ to 3’ direction.
  • the spacer sequence precedes the repeat sequence in a 5’ to 3’ direction.
  • the spacer(s) and repeat sequence(s) are linked directly to one another.
  • a linker is present between the spacerfs) and repeat sequence(s). Linkers may be any suitable linker.
  • a crRNA is useful as a single nucleic acid system for compositions, methods, and systems described herein or as part of a single nucleic acid system for compositions, methods, and systems described herein. In some embodiments, a crRNA is useful as part of a single nucleic acid system for compositions, methods, and systems described herein.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA wherein, a repeat sequence of a crRNA is capable of connecting a crRN A to an effector protein.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA linked to another nucleotide sequence that is capable of being non-covalently bond by an effector protein.
  • a repeat sequence of a crRNA can be linked to an intermediary RNA.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA and an intermediary RNA.
  • a crRNA is sufficient to form complex with an effector protein
  • the repeat sequence in the crRNA polynucleotide hybridizes with a tracr sequence present in a separate polynucleotide. In some embodiments, the hybridization with the tracr sequences permits formation of an RNP complex with an effector protein
  • a crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a crRNA comprises about: 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27. 28. 29. 30. 31. 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. 53. 54. 55. 56. 57. 58. 59. or 60 linked nucleotides.
  • a crRNA comprises at least: 10, 15, 20, 25, 30,
  • the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45. about 50, about 55. about 60, about 65, about 70 or about 75 linked nucleotides. sgRNA
  • a guide nucleic acid comprises a single guide RNA (sgRNA).
  • the guide nucleic acid is a sgRNA.
  • the combination of a spacer sequence e.g.. a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid
  • a handle sequence may be referred to herein as a single guide RNA (sgRNA), wherein the spacer sequence and the handle sequence are covalently linked.
  • the spacer sequence and handle sequence are linked by a phosphodiester bond.
  • the spacer sequence and handle sequence are linked by one or more linked nucleotides.
  • a guide nucleic acid may comprise a spacer sequence, a repeat sequence, or handle sequence, or a combination thereof.
  • the handle sequence may comprise a portion of, or all of, a repeat sequence.
  • a sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence.
  • compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g., a single nucleic acid system), wherein the guide RNA is a sgRNA.
  • a sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and/or modification activity of an effector protein on a target nucleic acid (e.g.. a hairpin region).
  • a target nucleic acid e.g.. a hairpin region
  • a sgRNA comprises one or more of one or more of a handle sequence, an intermediary' sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof.
  • a sgRNA comprises a handle sequence and a spacer sequence: an intermediary sequence and an crRNA; an intermediary sequence, a repeat sequence and a spacer sequence, and the like.
  • a sgRNA comprises an intermediary sequence and an crRNA.
  • an intermediary sequence is 5' to a crRNA in an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and crRNA.
  • an intermediary sequence and a crRNA are linked in an sgRNA directly (e g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises a handle sequence and a spacer sequence.
  • a handle sequence is 5’ to a spacer sequence in an sgRNA.
  • a sgRNA comprises a. linked handle sequence and spacer sequence.
  • a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • a handle sequence and a spacer sequence are finked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises an intermediary' sequence, a repeat sequence, and a spacer sequence.
  • an intermediary sequence is 5' to a repeat sequence in an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and repeat sequence
  • an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermedian- sequence and a repeal sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a repeat sequence is 5' to a spacer sequence in an sgRNA.
  • a sgRNA comprises a linked repeat sequence and spacer sequence.
  • a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodi ester bond)
  • a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • An exemplary’ handle sequence in a sgRNA may comprise, from 5‘ to 3’, a 5‘ region, a hairpin region, and a 3’ region.
  • the 5’ region may hybridize to the 3’ region.
  • the 5’ region does not hybridize to the 3’ region.
  • the 3" region is covalently linked to a spacer sequence (e.g.. through a phosphodiester bond)
  • the 5’ region is covalently linked to a spacer sequence (e.g. , through a phosphodiester bond).
  • compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid.
  • the effector protein is transactivated by the tracrRNA.
  • acti vity of the effector protein requires binding to a tracrRNA molecule.
  • a repeat hybridization sequence is at the 3’ end of a tracrRNA.
  • a repeat hybridization sequence may have a length of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8. about 9. about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides.
  • the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.
  • systems, compositions, and methods comprise a crRNA or a use thereof.
  • a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence.
  • the repeat sequence and the spacer sequences are directly connected to each other (e g.. covalent bond (phosphodi ester bond)).
  • the repeat sequence and the spacer sequence are connected by a linker.
  • systems, compositions, and methods comprise a tracrRNA or a use thereof. In some embodiments, systems, compositions, and methods do not comprise a tracrRNA or a use thereof, A tracrRNA and/or tracrRNA-crRN A duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRN A.
  • the secondary structure modifies activity of the effector protein on a target nucleic acid.
  • the secondary structure comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • the secondary structure comprises a pseudoknot (e g., a secondary structure comprising a stem at least partially hybridized to a second stem or halfstem secondary structure).
  • An effector protein may recognize a secondary 7 structure comprising multiple stem regions.
  • nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary' structure comprises one or more loops. In some embodiments, the secondary 7 structure comprises at least one, at least two, at least three, at least four, or at least five loops.
  • the present disclosure provides guide nucleic acids for use in combination with the effector proteins, RDDPs, and fusion proteins thereof described herein for precision editing of a target nucleic acids sequence.
  • guide nucleic acids for use in precision editing comprise a spacer sequence, a repeat sequence, a primer binding sequence, and a template sequence.
  • guide nucleic acids for use in precision editing comprise one or more linkers between one or more components of the guide nucleic acids.
  • a spacer sequence, a repeat sequence, a primer binding sequence, and a template sequence are comprised in a single polynucleotide, referred to herein as an extended guide RNA (rtgRNA).
  • a spacer sequence, a repeat sequence, a primer binding sequence, and a template sequence are comprised in two polynucleotides - the spacer and repeat sequence comprised in a first polynucleotide and the primer binding sequence and the template sequence comprised in a second polynucleotide, referred to herein as a split RNA. See e.g , FIG. 5A, FIG. 5B, and FIG. 6.
  • compositions, systems, and methods described herein comprise a template RNA (retRNA), wherein the template RNA (retRN A ) comprises a primer binding sequence and a template sequence.
  • the template RNA (retRNA) may also be referred to as an extension of a guide RNA.
  • the extension may comprise 1 , 2, 3. 4, 5, 6. 7, 8, 9. 10, 1 1 , 12, 13, 14. 15. 16, 17, 18, 19, or 20 nucleotides.
  • the extension may comprise more than 10 nucleotides, in some embodiments the extension is 10-20 nucleotides. In some embodiments the extension is 20-30 nucleotides. In some embodiments the extension is 30-40 nucleotides. In some embodiments the extension is 40-50 nucleotides.
  • the extension is 50-60 nucleotides. In some embodiments the extension is 70-80 nucleotides. In some embodiments the extension is 80-90 nucleotides. In some embodiments the extension is 90-100 nucleotides. In some embodiments the extension is 100-150 nucleotides.
  • the extension may be processed during guide RNA formation.
  • the retRNA, the spacer sequence, and the repeat sequence are comprised in the same polynucleotide (e.g, an rtgRNA). In some embodiments, the spacer sequence and repeat sequence are comprised in a first polynucleotide and the retRNA is comprised in a second polynucleotide (e g., a split RNA system).
  • the primer binding sequence hybridizes to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the target dsDNA molecule.
  • the spacer sequence is complementary 7 to the target sequence on the target strand of the dsDNA molecule, and the primer binding sequence and/or the template sequence is complementary to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the spacer sequence is complementary' to the target sequence on the non-target strand of the dsDNA molecule, and the primer binding sequence and'' or the template sequence is complementary to a primer sequence on the target strand of the target dsDNA molecule.
  • the primer binding sequence is 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long.
  • the template sequence is at least 10, 1 1 , 12. 13. 14, 15, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25. 26. 27, 28, 29, 30. 31 , 32, 33,
  • the template sequence may comprise one or more nucleotides having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when a spacer sequence of the guide RNA and the target sequence are aligned for maximum identity.
  • the one or more nucleotides may be contiguous.
  • the one or more nucleotides may not be contiguous.
  • the one or more nucleotides may each independently be selected from guanine, adenine, cytosine and thymine.
  • rtgRNA Extended guide RNA
  • the present disclosure provides extended guide nucleic acids (rtgRNA) and an effector protein and RDDP, or fusion protein thereof described herein, or nucleic acids encoding the same, wherein the spacer sequence, repeat sequence, template sequence, and primer binding sequence are each comprised in a single polynucleotide.
  • rtgRNA extended guide nucleic acids
  • the rtgRNA comprises, from 5‘ to 3’, a template sequence, a primer binding sequence, a repeat sequence, and a spacer sequence, optionally wherein a linker sequence is located between the primer binding sequence and the repeat sequence. See e.g, FIG. 3.
  • the rtgRNA comprises, from 5’ to 3’, a repeat sequence, a spacer sequence, a template sequence, and a primer binding sequence, optionally wherein a linker sequence is located between the template sequence and the spacer sequence. See e.g., FIG. 4.
  • the present disclosure provides a split gRNA system, comprising a first polynucleotide comprising a spacer sequence and a repeat sequence (e.g., a gRNA) and a second polynucleotide comprising a primer binding sequence and a template sequence (e.g., an retRNA), and an effector protein and RDDP. or fusion protein thereof described herein, or nucleic acids encoding the same.
  • the first polynucleotide comprises a spacer sequence and a repeat sequence.
  • the first polynucleotide is a crRNA, as described above.
  • the first polynucleotide comprises a spacer sequence and a handle sequence (also referred to herein as a scaffold sequence).
  • the first polynucleotide is an sgRNA, as described above.
  • the second polynucleotide comprises a primer binding sequence and a template sequence (e.g., an retRNA).
  • the second polynucleotide further comprises an aptamer that is recognized by a biological tether protein linked to an RDDP described herein.
  • the aptamer is an MS2 aptamer (See Said et al (November 2009). "In vivo expression and purification of aptamer-tagged small RNA regulators". Nucleic Acids Research. 37 (20): el 33; and Johansson et al (1997). "RNA recognition by the MS2 phage coat protein". Seminars in Virology. 8 (3): 176-185).
  • the second polynucleotide comprises, from 5" to 3". an aptamer sequence, a template sequence, and a primer binding sequence See FIG. 5B. In some embodiments, the second polynucleotide comprises, from 5’ to 3’, template sequence, a primer binding sequence, and an aptamer sequence. See FIG. 5A. In FIG. SA and FIG. SB, an MS2 aptamer is shown on the 3’ and 5' ends of the retRNA. respectively. The MS2 aptamer is an exemplary sequence and an RNA sequence with the appropriate secondary structure may also be used instead of the aptamer. [0290] In some embodiments, the second polynucleotide is circularized. See FIG. 6.
  • compositions, systems and methods for modifying a target nucleic acid wherein the target nucleic acid is a gene, a portion thereof a transcript thereof.
  • the target nucleic acid is a double stranded nucleic acid.
  • the double stranded nucleic acid is DNA.
  • die target nucleic acid comprises a eukaryotic gene.
  • the target nucleic acid comprises a human gene.
  • the target nucleic acid comprises an intron of a gene.
  • the target nucleic acid comprises a sequence that does not encode a protein (also referred to as a non-coding region).
  • the target nucleic acid comprises a safe harbor locus.
  • a “safe harbor locus” refers to a region of the genome that can maintain transgene expression without detrimentally altering the function of host ceils. Safe harbor loci are known in the art and include, but are not limited to AAVSI (FPPIR12C) gene, an ALB gene, an ANGPTL3 gene, an APOC3 gene, an ASGR2 gene, a CCR5 gene, a FIX(F9)‘ gene, a G6PC gene, a Gys2 gene, an HGD gene, &LP(A) gene, a PCSK9 gene, a SERPINA1 gene, a TF gene, a TTR gene, ROGI1, ROGI2, and an intron thereof (See e.g., Sadelain, M. dislike Papapetrou, E.P., and Bushman, ED. (2012). Safe harbours for the integration of new DNA in the human genome. Nat. Rev. Cancer 12, 51-58;
  • an LSR or system comprising the same described herein are used to insert a donor nucleic acid sequence encoding a wildtype or functional protein (or functional domain thereof) into a safe harbor locus.
  • the donor nucleic acid encodes a wildtype or functional version of any one of the genes listed in TABLE 10.
  • an LSR or system comprising the same described herein is used to disrupt a gam of function allele or an allele encoding a misfolded protein.
  • the target nucleic acid is a double stranded nucleic acid comprising a target strand find a non-target strand, wherein the target strand comprises a target sequence.
  • a target strand comprises a target sequence
  • at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand.
  • the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand
  • the target strand comprises a target sequence
  • at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand.
  • a target nucleic acid comprises a PAM as described herein that is located on the non-target strand.
  • a PAM described herein in some embodiments, is adjacent (e.g., within 1 , 2, 3, 4. 5, 10, 20, 25 nucleotides) to the 5’ or 3’ end of the target sequence on the non-target strand of the double stranded DNA molecule.
  • such a PAM described herein is directly adjacent to the 5’or 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule.
  • an effector protein described herein, or a multimeric complex thereof recognizes a PAM on a target nucleic acid.
  • multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid.
  • only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid.
  • the PAM is 3’ to the spacer region of the crRNA.
  • the PAM is directly or adjacent (e.g., within 1 , 2, 3, 4, 5, 10, 20, 25 nucleotides) 3’ or 5’ to the spacer region of the crRNA.
  • the PAM comprises a PAM sequence set forth in TABLE 4.
  • An effector protein or fusion protein of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • PAM protospacer adjacent motif
  • cleavage occurs within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides of a 5 ’ or 3 ’ terminus of a PAM sequence.
  • a target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.
  • the target nucleic acid is in a cell.
  • the cell is a single-cell eukaryotic organism; a plant cell; an algal cell; a fungal cell; an animal cell; a cell of an invertebrate animal; a cell of a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a ceil of a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and caprine.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell, a human cell, or a plant cell.
  • the cell is a human ceil.
  • compositions, systems and methods disclosed herein may be useful for treating a disease in a subject by inserting a donor nucleic acid (transgene) into the genome utilizing an LSR system described herein, wherein the insertion results in production of a functional protein
  • LOF loss of function
  • Non-limiting examples of LOF diseases are spinal muscular atrophy (SMA), Pompe disease, Duchene muscular dystrophy. Fanconi -Bickel syndrome, Danon disease, von Gierke's disease, Cori's disease, Forbes' disease, McArdle's disease, Hers' disease, Tarin's disease, Fanconi-Bickel syndrome.
  • Aldolase A deficiency cystic fibrosis, hemophilia A, hemophilia B, Giteiman syndrome.
  • Tay-Sachs disease sickle ceil anemia, familial hypercholesterolemia, hemochromatosis, Huntington’s disease, nephrogenic diabetes insipidus, familial glucocorticoid deficiency, familial hypogonadism, hypogonadism, euthyroid hyperthyroidism, central hypothyroidism, chondrodysplasia, benign familial hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism, retinal degeneration, vitreoretinopathy , Hirschsprung disease, fragil e X syndrome, Gaucher’s Disease, glucose 6-phosphate dehydrogenase deficiency, holoprosencephaly, phenylketonuria, Friedreich’s ataxia, alpha- 1 antitrypsin deficiency.
  • the methods comprise administration of an LSR system, or nucleic acid encoding the same, or composition comprising the same to a subject in need thereof.
  • methods comprise administering a cell that has been modified by a system described herein to a subject.
  • the disease may be a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof.
  • the disease may be an inherited disorder, also referred to as a genetic disorder.
  • a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or target sequence.
  • the term donor nucleic acid refers to a sequence of nucleotides that will be or has been introduced into a cell following transfection of the viral vector.
  • the donor nucleic acid may be introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to. integration into the genome of the cell or introduction of an episomal plasmid or viral genome via an integration sequence and an LSR.
  • the term donor nucleic acid refers to a sequence of nucleotides that will be.
  • Donor nucleic acids of any suitable size may be integrated into a target nucleic acid or genome.
  • the donor polynucleotide integrated into a genome is less than 3, about 3, 3.5, 4, 4.5, 5. 5.5. 6, 6.5, 7, 7.5, 8, 8.5. 9, 9.5. 10, 10.5, 11. 11.5, 12. 12.5, 13, 13.5, 14, 14 5, 15, 16, 17, 1 8, 19, 20, 25, 30. 35, 40, 45, 50, 100, 150. 200, 250, 300, 350, 400, 450, 500 kilobases in length.
  • donor nucleic acids are more than 500 kilobases (kb) in length.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the SMN protein, or a mutation in the SMNI gene, in a subject with spinal muscular atrophy (SAIA).
  • SAIA spinal muscular atrophy
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the acid alpha-glucosidase protein, or a mutation in the GAA gene, in a subject with Pompe disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the dystrophin protein, or a mutation in the DMD gene, in a subject with Duchene muscular dystrophy.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the GI..UT2 protein, or a mutation in the SLC2A2 gene, in a subject with Fanconi -Bickel syndrome.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the LAMP2 protein, or a mutation in the Mac-3 gene, in a subject with Danon disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glucose-6-phosphatase protein, a mutation in the G6PC gene, a mutation in the glucose-6-phosphate transporter, a mutation in the SLC37A4 gene, a mutation in the SLC17A3 protein, or a mutation in the SLC17A3 gene in a subject with von Gierke's disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glycogen debranching enzyme, or a mutation in the zlG'L gene, in a subject with Cori's disease or Forbes' disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glycogen phosphorylase protein, or a mutation in the PYGM gene, in a subject with McArdle's disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of the function due to a mutation in the glycogen phosphorylase protein, or a mutation in the PYGL gene, in a subject with Hers' disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the GLUT2 protein, or a mutation in the SLC2A2 gene, in a subject with Fanconi-Bickel syndrome.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the fructose-bisphosphate aldolase protein, or a mutation in the ALDOA gene, in a subject with Aldolase A deficiency .
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the factor IX protein, or a mutation in the F9 gene, in a subject with hemophilia B.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the sodium chloride cotransporter protein, a mutation in the SLC12A3 gene, a mutation in the chloride voltage-gated channel Kb protein, or a mutation in the CLCNKB gene in a subject with Gitelnian syndrome.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the hemoglobin subunit beta protein, or a mutation in the HBB gene, in a subject with sickle cell anemia.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the LDL receptor protein, a mutation in the LDLR gene, a mutation in the apolipoprotein B protein, a mutation in the APOB gene.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Huntingtin protein, or a mutation in the HIT gene, in a subject with Huntington's disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Vasopressin receptor 2 (V2R), a mutation in the A KPAG gene, a mutation in the Aquaporin-2 protein, or a mutation in the A QP2 gene, in a subject with nephrogenic diabetes insipidus.
  • V2R Vasopressin receptor 2
  • a KPAG a mutation in the A KPAG gene
  • Aquaporin-2 protein a mutation in the Aquaporin-2 protein
  • a QP2 a mutation in the A QP2 gene
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the melanocortin 2 receptor protein, a mutation in the MC2R gene, a mutation in the melanocortin 2 receptor accessory protein, a mutation in the MRAP gene, a mutation in the nicotinamide nucleotide transhydrogenase protein, or a mutation in the NNT gene, in a subject with familial glucocorticoid deficiency.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function on due to a mutation in the KiSS l-derived peptide receptor protein, or a mutation in the KISSI gene, in a subject with familial hypogonadism.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the thy rotropin receptor protein, or a mutation in the TSHR gene, in a subject with euthyroid hyperthyroidism
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the immunoglobulin superfamily, member 1 protein, or a mutation in the IGSF1 gene, in a subject with central hypothyroidism.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the collagen ty pe II alpha 1 chain protein, a mutation in the COL2A / gene, a mutation in the fibroblast growth factor receptor 3 protein, a mutation in the FGFR3 gene, a mutation in the sulfate transporter protein, or a mutation in the SLC26A2 gene, in a subject with chondrodysplasia.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the calcium-sensing receptor protein, or a mutation in the CASR gene, in a subject with benign familial hypocalciunc hypercalcemia or neonatal severe primary' hyperparathyroidism.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the rhodopsin protein, or a mutation in the RHO gene, in a subject with retinal degeneration.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the frizzled class receptor 4 protein, a mutation in the FZD4 gene, a mutation in the LDL receptor related protein 5 protein, a mutation in the LRP5 gene, a mutation in the norrin cystine knot growth factor protein, or a mutation in the NDP gene, in a subject with vitreoretinopathy.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the glucosylceramidase beta 1 protein, or a mutation in the GBA1 gene, in a subject with Gaucher's Disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Glucose-6-phosphate dehydrogenase protein, or a mutation in the G6PD gene, in a subject with glucose 6-phosphate dehydrogenase deficiency.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the Glucose-6-phosphate dehydrogenase protein, or a mutation in the G6PD gene, in a subject with glucose 6-phosphate dehydrogenase deficiency.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the SIX hoineobox 3 protein, or a mutation in the SIX3 gene, m a subject with holoprosencephaly.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the phenylalanine hydroxylase protein, or a mutation in the PAH gene, in a subject with phenylketonuria.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the frataxin protein, or a mutation in the FXN gene, in a subject wi th F ri edrei ch ’ s ataxi a.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the serpin family A member 1 protein, or a mutation in the SERPINA1 gene, in a subject with alpha- 1 antitrypsin deficiency.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the sphingomyelin phosphodiesterase 1 protein, a mutation in the SMPD! gene, a mutation in the NPC intracellular cholesterol transporter 1 protein, a mutation in ths NPC1 gene, a mutation in ths NPC intracellular cholesterol transporter 2 protein, or a mutation in the NPC2 gene, in a subject with Niemann Pick's disease.
  • the donor nucleic acid replaces a nucleic acid that causes a loss of function due to a mutation in the poly cystin 1 protein, a mutation in the PKD1 gene, a mutation in the polycystin 2 protein, a mutation in the PKD2 gene, a mutation in the PKHD1 ciliary 7 1PT domain containing fibrocystin/polyductin protein, or a mutation in the PKHDi gene, in a subject with polycystic kidney disease.
  • Polypeptides ( ⁇ ?.g.. effector proteins, LSRs. and/or RDDPs) and nucleic acids (e.g.. engineered guide nucleic acids) described herein can be further modified as described throughout and as further described herein. Examples are modifications of interest that do not alter primary' sequence, including chemical denvatization of polypeptides, e.g, acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.
  • modifications of interest that do not alter primary' sequence, including chemical denvatization of polypeptides, e.g, acylation, acetylation, carboxylation, amidation, etc.
  • sequences that have phosphorylated amino acid residues e.g. phosphotyrosine, phosphoserine, or phosphothreonine.
  • Modifications disclosed herein can also include modif ication of described polypeptides and/or engineered guide nucleic acids through any suitable method, such as molecular biological techniques and/or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity', enzymatic activity, etc.) or to render them more suitable.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
  • Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. [0348] Modifications can further include the introduction of various groups to polypeptides and/or engineered guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g, an effector protein, LSR, base editing enzyme, and/or RDDP). which allow for linking to other molecules or to a surface.
  • a polypeptide e.g, an effector protein, LSR, base editing enzyme, and/or RDDP
  • cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
  • Modifications can further include modification of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature.
  • modifications of a nucleic acid include a base modification, a backbone modification, a sugar modification, or combinations thereof, of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.
  • nucleic acids e.g.. engineered guide nucleic acids
  • nucleic acids described herein comprise one or more modifications comprising: 2’O-methyl modified nucleotides. 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g, a 7-methylguanylate cap (m7G)).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • phosphorothioates chiral phosphoro thioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3‘-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoraniidates including 3'-amino phosphoramidate and aminoalkyl phosphoraniidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates .
  • thionoalkylphosphotriesters having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5’ to 5’ or 2' to 2' linkage; phosphorothioate and/or heteroatom intern ucleoside linkages, such as -CH2-NH-O-
  • CH2-, -CH2-N(CHa)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH?- O-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )- N(CH 3 )-CH 2 - and -O-NK’H 3 )-CH 2 -CH 2 - (wherein the native phosphodiester internucleotide linkage is represented as -O-P("O)(OH)-O-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbone
  • compositions and systems provided herein comprise a vector system encoding a polypeptide (e.g. , an LSR, effector protein, base editing enzy me, an RDDP, and/or a fusion protein thereof) and/or a guide nucleic acid described herein.
  • compositions and systems provided herein comprise a vector system encoding a guide nucleic acid (e.g. , crRNA) described herein.
  • compositions and systems provided herein comprise a multi-vector system encoding an LSR, effector protein, an RDDP, and/or a fusion protein thereof and a guide nucleic acid described herein, wherein the guide nucleic acid and the LSR, effector protein, the RDDP, and/or the fusion protein thereof are encoded by the same or different vectors.
  • the guide and the engineered effector protein are encoded by different vectors of the system.
  • a nucleic acid encoding a polypeptide e.g , an LSR, an effector protein, base editing enzyme, an RDDP, and/or a fusion protein thereof
  • a nucleic acid encoding a polypeptide is a messenger RNA.
  • an expression vector comprises or encodes an engineered guide nucleic acid.
  • the expression vector encodes the crRNA
  • a vector can comprise or encode one or more regulatory elements. Regulatory elements can refer to transcriptional and translational control sequences, such as promoters, enhancers, poly adenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide.
  • a vector can comprise or encode for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like.
  • Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3' direction) coding or non-coding sequence.
  • a promoter can be bound at its 3' terminus to a nucleic acid the expression or transcription of which is desired, and extends upstream (5' direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level.
  • a promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”.
  • a promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of' transcription machinery, such as RNA polymerase.
  • promoters When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes.
  • Various promoters, including inducible promoters, may be used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.
  • Promotors can be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g. , CMV promoter), inducible promoters (e.g. , heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc,), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc,), etc.
  • constitutively active promoters e.g. , CMV promoter
  • inducible promoters e.g. , heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc,
  • spatially restricted and/or temporally restricted promoters e.g., a tissue specific promoter, a cell type specific promoter, etc, etc.
  • Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl).
  • SV40 early promoter mouse mammary tumor virus long terminal repeat (LTR) promoter
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • Hl human Hl promoter
  • vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and/or a nucleic acid that encodes an effector protein io a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and/or an effector protein
  • vectors provided herein comprise at least one premotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein.
  • the viral vector comprises a nucleotide sequence of a promoter.
  • the viral vector comprises two promoters.
  • the viral vector comprises three promoters.
  • the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides.
  • the length of the promoter is at least 100 linked nucleotides.
  • Non-limiting examples of promoters include CMV, 7SK. EFla, RPBSA, hPGK, EFS.
  • the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide.
  • Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracyclme- repressibie), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter.
  • the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al.. (2019).
  • the promoter for expressing effector protein is a muscle-specific promoter.
  • the muscle-specific promoter comprises Ck8e, SPC5-12, or Desmin promoter sequence.
  • the promoter for expressing effector protein is a ubiquitous promoter.
  • the ubiquitous promoter comprises MND or CAG promoter sequence.
  • an effector protein, base editing enzyme, LSR, RDDP, or fusion protein comprising the same (or a nucleic acid encoding same) and/or a guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are co-administered with a donor nucleic acid.
  • Coadininistration can be contact with a target nucleic acid, administered to a ceil, such as a host cell, or administered as method of nucleic acid detection, editing, an d/'or treatment as described herein, in a single vehicle, such as a single expression vector.
  • an effector protein, base editing enzyme LSR, RDDP, or fusion protein comprising the same (or a nucleic acid encoding same) and/or agui de nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single v ehicle.
  • an effector protein (or a nucleic acid encoding same), a guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and/or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or tw’O or more expression vectors.
  • An expression vector can be a viral vector.
  • a viral vector comprises a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle.
  • the nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented.
  • the nucleic acid may comprise DNA, RNA, or a combination thereof.
  • the expression vector is an adeno-associated viral vector.
  • viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • retroviruses e g., lentiviruses and y-retroviruses
  • adenoviruses e g., lentiviruses and y-retroviruses
  • AAVs adeno-associated viruses
  • baculoviruses baculoviruses
  • vaccinia viruses herpes simplex viruses and poxviruses.
  • a viral vector provided herein can be derived from or based on any such virus.
  • the viral vectors provided herein are an adeno-associated viral vector
  • an AAV vector has two inverted terminal repeats (ITRs).
  • the viral vector provided herein comprises two inverted terminal repeats of AAV.
  • the DNA sequence in between the ITRs of an AAV vector provided herein may be referred to herein as the sequence encoding the genome editing tools or a transgene.
  • These genome editing tools can include, but are not limited to, an effector protein, effector protein modifications (c.g, nuclear localization signal (NLS), poly A tail), guide nucleic acid(s), respective promoter(s), and a donor nucleic acid, or combinations thereof.
  • a nuclear localization signal comprises an entity' (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.
  • viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein.
  • the length of the promoter is less than about 500, less than about 400. or less than about 300 linked nucleotides In some embodiments, the length of the promoter is at least 100 linked nucleotides.
  • promoters include
  • the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide.
  • Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline- repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter
  • the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzm el al.
  • the coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating an AAV vector that is a self-complementary AAV (scAAV) vector.
  • scAAV self-complementary AAV
  • the sequence encoding the genome editing tools of an scAAV vector has a length of about 2 kb to about 3 kb.
  • the scAAV vector can comprise nucleotide sequences encoding an effector protein, providing guide nucleic acids described herein, and a donor nucleic acid described herein.
  • the AAV vector provided herein is a self-inactivating AAV vector.
  • an AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector
  • the viral particle that delivers the viral vector described herein is an AAV.
  • AAVs are characterized by their serotype.
  • Non-limiting examples of AAV serotypes are AAV1.
  • the AAV particles described herein can be referred to as recombinant AAV (rAAV).
  • rAAV particles are generated by transfecting AAV producing cells with an AAV- containing plasmid earn ing the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, l.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as EIA, E1B, E2A, E4ORF6 and VA.
  • the AAV producing ceils are mammalian cells.
  • host cells for rAAV viral particle production are mammalian ceils.
  • a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a derivative thereof, or a combination thereof.
  • rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell.
  • producing rAAV virus particles in a mammalian cell can comprise transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3' ends.
  • production of rAAV virus particles in insect cells can comprise infecting the insect cells with three recombinant baculoviruses. one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end.
  • rAAV virus particles are produced by the One Bac system.
  • rAAV virus particles can be produced by the Two Bac system.
  • the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome.
  • an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example.
  • compositions and systems provided herein comprise a lipid particle.
  • a lipid panicle is a lipid nanoparticle (LNP).
  • a lipid or a lipid nanoparticle can encapsulate an expression vector.
  • a lipid or a lipid nanoparticle can encapsulate the effector protein, the LSR, the RDDP, the sgRNA or crRNA, the nucleic acid encoding the effector protein, or LSR, or RDDPand/or the DNA molecule encoding the sgRNA or crRNA.
  • LNPs are effective for delivery of nucleic acids.
  • a method can comprise contacting a cell with an expression vector.
  • contacting can comprise electroporation, lipofection, or lipid nanoparticle (LNP) delivery of an expression vector.
  • a guide nucleic acid (or a nucleic acid comprising a nucleotide sequence encoding same), an LSR, an RDDP, and/or an effector protein described herein can be introduced into a host cell by any of a variety of well-known methods
  • a guide nucleic acid, an LSR. an RDDP, and/or effector protein can be combined with a lipid.
  • a guide nucleic acid, an LSR, an RDDP and/or effector protein can be combined with a particle or formulated into a particle.
  • Methods of editing described herein may be employed to generate a genetically modified cell.
  • the cell may be a eukaiyotic cell (e.g., a mammalian cell) or a prokaryotic ceil (e.g., an archaeal cell).
  • the cell may be derived from a multicellular organism and cultured as a unicellular entity.
  • the cell may comprise a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation.
  • the cell may be progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell.
  • a genetically modified cell may comprise a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived.
  • the present disclosure provides a mammalian cell comprising a system described herein, e.g, an RDDP described herein (or a fusion protein comprising the same), an effector protein described herein (or a fusion protein comprising the same), an LSR (or a fusion protein comprising the same), a base editing enzyme (or fusion protein comprising the same) and/or an rtgRNA.
  • a system described herein e.g, an RDDP described herein (or a fusion protein comprising the same), an effector protein described herein (or a fusion protein comprising the same), an LSR (or a fusion protein comprising the same), a base editing enzyme (or fusion protein comprising the same) and/or an rtgRNA.
  • the present disclosure provides a mammalian cell comprising one or more polynucleotides encoding one or more components of a system described herein, or one or more vectors comprising the same, e.g., a polynucleotide encoding an RDDP, base editing enzyme, or LSR described herein (or a fusion protein comprising the same) or vector comprising the same, a polynucleotide encoding an effector protein described herein (or a fusion protein comprising the same) or a vector comprising the same, and/or a polynucleotide encoding an rtgRNA or a vector comprising the same.
  • a mammalian cell comprising one or more polynucleotides encoding one or more components of a system described herein, or one or more vectors comprising the same, e.g., a polynucleotide encoding an RDDP, base editing enzyme, or LSR described herein (or
  • compositions of the disclosure may be administered to a subject.
  • a subject may be a human.
  • a subject may be a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse).
  • a subject may be a vertebrate or an invertebrate.
  • a subject may be a laboratory animal.
  • a subject may be a patient.
  • a subject may be at risk of developing, suffering from, or displaying symptoms a disease or disorder as set forth in herein.
  • a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • a point mutation optionally comprises a substitution, insertion, or deletion.
  • a mutation comprises a chromosomal mutation.
  • a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation.
  • a mutation comprises a copy number variation.
  • a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat.
  • a cell may be in vitro.
  • a cell may be in vivo.
  • a cell may be ex vivo.
  • a cell may be an isolated cell.
  • a cell may be a ceil inside of an organism.
  • a ceil may be an organism.
  • a cell may be a cell m a cell culture
  • a cell may be one of a collection of cells.
  • a cell may be a mammalian cell or derived from a mammalian cell.
  • a cell may be a rodent cell or derived from a rodent ceil.
  • a cell may be a human cell or derived from a human cell.
  • a cell may be a eukatyotic cell or derived from a eukatyotic cell.
  • a cell may be a pluripotent stem cell.
  • a cell may be a plant cell or derived from a plant cell.
  • a cell may be an animal cell or derived from an animal cell.
  • a cell may be an invertebrate cell or derived from an invertebrate cell.
  • a cell may be a vertebrate cell or derived from a vertebrate cell.
  • a cell may be from a specific organ or tissue.
  • Non-limiting examples of organs and tissues from which a cell may be obtained or in which a cell may be located include: muscle, adipose, bone, adrenal gland, pituitary gland, thyroid gland, pancreas, testes, ovaries, uterus. heart, lung, aorta, smooth vasculature, endometrium, brain, neurons, spinal cord, kidney, liver, esophagus, stomach, intestine, colon, bladder, and spleen.
  • the tissue may be the subject's blood, bone marrow, or cord blood.
  • the tissue may be heterologous donor blood, cord blood, or bone marrow.
  • the tissue may be allogenic blood, cord blood, or bone marrow.
  • the cell is a stem cell
  • stem cells are hematopoietic stem cells, muscle stem cells (also referred to as myoblasts or muscle progenitor cells), and pluripotent stem cells (including induced pluripotent stem cells).
  • the cell is cell derived or differentiated from a pluripotent stem cell
  • the cell is a hepatocyte.
  • the cell is an immune cell.
  • immune cells are lymphocytes (T cells, B cells, and NK cells), neutrophils, and monocytes/ macrophages.
  • compositions for modifying a target nucleic acid in a cell or a subject comprising any one of the effector proteins, LSRs, RDDPs, fusion proteins thereof, or guide nucleic acids as described herein and any combination thereof.
  • pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, LSRs, RDDPs, fusion proteins thereof, or guide nucleic acids as described herein and any combination thereof.
  • pharmaceutical compositions comprise a plurality of guide nucleic acids
  • Pharmaceutical compositions may be used to modify 7 a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.
  • compositions comprising a viral vector encoding a effector proteins. LSRs. RDDPs. fusion proteins thereof and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein.
  • pharmaceutical compositions comprise a virus comprising a viral vector encoding an effector protein, LSR RDDP, fusion protein thereof, a guide nucleic acid, or a combination thereof: and a pharmaceutically acceptable earner or diluent.
  • the virus may be a lentivirus.
  • the virus may be an adenovirus.
  • the virus may be a non-replicating virus.
  • the virus may be an adeno-associated virus (AAV).
  • the viral vector may be a retroviral vector.
  • Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency vims (HIV) genome.
  • the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses
  • the viral vector is a recombinant viral vector.
  • the viral vector is an AAV
  • the AAV may be any AAV known in the art.
  • the viral vector corresponds to a virus of a specific serotype.
  • the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV 10 serotype, an AAV 11 serotype, and an AAV12 serotype.
  • the .AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof.
  • scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.
  • methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid, or transgene that, when transcribed, produces a guide nucleic acid; at least one nucleic acid that encodes: (i) a Replication (Rep) gene; and (it) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging a Cas effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector.
  • promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector.
  • the AAV vector comprises a sequence encoding a guide nucleic acid.
  • the guide nucleic acid comprises a crRNA.
  • the guide nucleic acid is a crRNA
  • the guide nucleic acid comprises a sgRNA.
  • the guide nucleic acid is a sgRNA.
  • the AAV vector can package 1, 2, 3, 4, or 5 nucleotide sequences encoding guide nucleic acids or copies thereof.
  • the AAV vector packages 1 or 2 nucleotide sequences encoding guide nucleic acids or copies thereof.
  • the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are the same. In some embodiments, the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are different. In some embodiments, the AAV vector comprises inverted terminal repeats, e g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat.
  • the inverted terminal repeat comprises inverted terminal repeats from AAV. In some embodiments, the inverted terminal repeat comprises inverted terminal repeats of ssAAV vector or scAAV vector. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.
  • a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (IT R) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same.
  • the Rep gene and ITR from a first A AV serotype e.g., AAV2
  • a second AAV serotype e.g , AAV 9
  • a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9.
  • the hybrid AAV delivery vector comprises an AAV2/I , A AV 2/2, AAV 2/4, AAV2/5. A AV 2/8. or AAV2/9 vector.
  • the AAV vector may be a chimeric AAV vector.
  • the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes
  • a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.
  • the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g. , a bacterial vector) a viral vector, or any combination thereof.
  • the delivery vehicle may be a non-viral vector.
  • the delivers' vehicle may be a plasmid.
  • the plasmid comprises DNA.
  • the plasmid comprises RNA.
  • the plasmid comprises circular double-stranded DNA.
  • the plasmid may be linear.
  • the plasmid comprises one or more genes of interest and one or more regulatory' elements
  • the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria.
  • the plasmid may be a minicircle plasmid
  • the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid.
  • the plasmid may be formulated for delivery through injection by a needle carrying syringe.
  • the plasmid may be formulated for delivery via electroporation.
  • the plasmids may be engineered through synthetic or other suitable means known in the art.
  • the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.
  • the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic ceil.
  • exemplary physical methods include electroporation, gene gun. sonoporation, magnetofection, or hydrodynamic delivery.
  • Exemplary' chemical methods include delivery’ of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparti cies; or cellpenetrating peptides [0384]
  • a fusion effector protein as described herein is inserted into a vector.
  • the vector comprises a transgene which comprises a nucleotide sequence of one or more promoters, enhancers, ribosome binding sues, RNA splice sites, polyadenylation sites, a replication origin, and/or transcriptional terminator sequences.
  • the AAV vector comprises a self-processing array system for guide nucleic acid.
  • a self-processing array system refers to a system for multiplexing, stringing together multiple guide nucleic acids under the control of a single promoter.
  • plasmids and vectors described herein comprise at least one promoter.
  • the promoters are constitutive promoters.
  • the promoters are inducible promoters.
  • the promoters are prokary otic promoters (e.g, drive expression of a gene in a prokaryotic cell).
  • the promoters are eukary otic promoters, (e.g...
  • promoters include, but are not limited to, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK promoter.
  • the promoter is CMV, In some embodiments, the promoter is EFla. In some embodiments, the promoter is U6. In some embodiments, the promote is Hl. In some embodiments, the promoter is 7SK.
  • the promoter is ubiquitin.
  • vectors are bicistronic or polycistronic vector (e g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.
  • IRS internal ribosome entry site
  • the AAV vector comprises a promoter for expressing effector proteins. LSRs, RDDPs. and/or fusion proteins thereof
  • the promoter for expressing effector proteins, LSRs, RDDPs, and/or fusion proteins thereof is a site-specific promoter.
  • the promoter for expressing effector proteins, LSRs, RDDPs, and/or fusion proteins thereof is a muscle-specific promoter.
  • the muscle-specific promoter comprises Ck8e, SPC5-12. or Desmin promoter sequence.
  • the promoter for expressing effector proteins, LSRs, RDDPs, and/or fusion proteins thereof is a ubiquitous promoter.
  • the ubiquitous promoter comprises MN D or CAG promoter sequence.
  • the 3' -untranslated region comprises a nucleotide sequence of an intron.
  • the 3 ’-untranslated region comprises one or more sequence elements, such as an intron sequence or an enhancer sequence.
  • the 3'- un translated region comprises an enhancer.
  • vectors comprise tin enhancer Enhancers are nucleotide sequences that have the effect of enhancing promoter activity.
  • enhancers augment transcription regardless of the orientation of their sequence.
  • enhancers activate transcription from a distance of several kilo basepairs.
  • enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription.
  • exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-LJ5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit 0-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3). p 1527-31. 1981); and the genome region of human growth hormone (J Immunol..
  • the enhancer is WPRE.
  • the AAV vector comprises one or more polyadenylation (poly A) signal sequences.
  • the polyadenlyation signal sequence comprises hGH poly A signal sequence.
  • the polyadenlyation signal sequence comprises sv40 poly A signal sequence.
  • compositions described herein may comprise a salt.
  • the salt is a sodium salt.
  • the salt is a potassium salt.
  • the salt is a magnesium salt.
  • the salt is NaCI.
  • the salt is KNO?.
  • the salt is Mg z+ SO4 2 ”.
  • Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g.
  • neutral buffered saline phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose, dextran, mannitol
  • polypeptides or ammo acids e.g, glycine
  • antioxidants e.g., EDTA. glutathione
  • adjuvants e.g. , aluminum hydroxide
  • surfactants Polysorbate 80, Polysorbate 20, or Plutonic F68
  • glycerol sorbitol; mannitol; polyethyleneglycol; and preservatives.
  • compositions are in the form of a solution (e.g., a liquid).
  • the solution may be formulated for injection, e.g, intravenous or subcutaneous injection.
  • the pH of the solution is about 7, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about. 8.5, about 8.6, about 8.7. about 8.8, about 8.9, or about 9.
  • the pH is 7 to 7.5, 7,5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5.
  • the pH of the solution is less than 7.
  • the pH is greater than 7.
  • compositions comprise an: effector protein, fusion effector protein, fusion partner, LSR, RDDP, base editing enzyme, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutical compositions comprise one or more nucleic acids encoding an: effector protein, fusion effector protein, fusion partner, LSR, RDDP, base editing enzyme, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides a system for the modification of a target nucleic acid comprising (1 ) a recombinase or a polynucleotide encoding the same and a donor DNA vector comprising a first recombinase recognition sequence (RRS-1) and wherein the target nucleic acid comprises a second RRS (RRS-2) sequence.
  • a target nucleic acid comprising (1 ) a recombinase or a polynucleotide encoding the same and a donor DNA vector comprising a first recombinase recognition sequence (RRS-1) and wherein the target nucleic acid comprises a second RRS (RRS-2) sequence.
  • the recombinase comprises an amino acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 1-3918 and 9761-9763
  • the RRS-1 sequence comprises a nucleic acid sequence that is at least 90%, al least, 91 %, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%. at least 98%.
  • the RRS-2 sequence comprises a nucleic acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase, RRS-2.
  • the recombinase comprises any one of SEQ ID NOs: 1-3918 and 9761- 9763
  • the RRS-1 comprises any one of SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791
  • the RRS-2 comprises any one of SEQ ID NOs: 3919-6441, 9719-9739. and 9792-9819, wherein the recombinase.
  • RRS-2 and RRS-2 are present in the same row in TABLE 1 or TABLE 2.
  • the recombinase consists of any one of SEQ ID NOs: 1- 3918 and 9761-9763
  • the RRS-1 consists of any one of SEQ ID NOs: 6442-8964, 9740-9760, and 9764-9791
  • the RRS-2 consists of any one of SEQ ID NOs: 3919-6441, 9719-9739, and 9792-9819, wherein the recombinase, RRS-2 and RRS-2 are present in the same row in TABLE I or TABLE 2.
  • the system further comprises an effector protein.
  • the effector protein sequence comprises an ammo acid sequence that is 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%. or at least 99% identical to any one of SEQ ID NOs: 9267- 9401 and 9698-9701.
  • the effector protein sequence comprises any one of SEQ ID NOs: 9267-9401 and 9698-9701.
  • the effector protein sequence consists of any one of SEQ ID NOs: 9267-9401 and 9698-9701.
  • the system further comprises an effector protein sequence and an RDDP sequence, or a fusion protein comprising an effector protein and an RDDP.
  • the RDDP sequence comprises an amino acid sequence that is 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%, or at least 99% identical to any one of SEQ ID NOs: 9402-9482
  • the effector protein sequence comprises an amino acid sequence that is at least 90%, at least, 9! %. at least 92%. at least 93%. at least 94%. at least 95%, at least 96%, at least 97%.
  • the RDDP sequence comprises any one of SEQ ID NOs: 9402-9482, and the effector protein sequence comprises any one of SEQ ID NOs: 9267-9401 and 9698-9701.
  • the RDDP amino acid sequence consists of any one of SEQ ID NOs: 9402-9482, and the effector protein amino acid sequence consists of any one of SEQ ID NOs: 9267-9401 and 9698-9701.
  • the RDDP-effector fusion comprises an amino acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9702-9713.
  • the RDDP-effector fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 9702-9713.
  • RDDP-effector fusion consists of any one of the sequences selected from SEQ ID NOs: 9702-9713.
  • the recombinase comprises an amino acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441 , 454, 455. 569, 591. 620, 1105. 1 121. 1319, 1452, 1518, 1944, 1966, 2058, 2068. 2172, and 9761-9763.
  • the RRS-I sequence comprises a nucleic acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9740-9791
  • the RRS-2 sequence comprises a nucleic acid sequence that is 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%, or at least 99% identical to any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819, wherein the recombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15.
  • the recombinase comprises any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441, 454, 455, 569, 591. 620, 1105, 1121, 1319, 1452, 1518, 1944, 1966, 2058, 2068, 2172, and 9761-9763
  • the RRS-1 sequence comprises any one of the sequences selected from SEQ ID NOs: 9740-9791
  • the RRS-2 sequence comprises any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819, wherein the recombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15.
  • the recombinase consists of any one of the sequences selected from SEQ ID NOs: 3, 16, 18, 20, 350, 441. 454, 455. 569, 591, 620, 1 105. 1121, 1319, 1452, 1518, 1944. 1966, 2058, 2068, 2172, and 9761-9763.
  • the RRS-1 sequence consists of any one of the sequences selected from SEQ ID NOs: 9740-9791
  • the RRS-2 sequence consists of any one of the sequences selected from SEQ ID NOs: 9719-9739 and 9793-9819. wherein the recombinase, the RRS-1 and RRS-2 sequences are on the same line in any of TABLES 13-15.
  • N is any’ nucleotide.
  • V is A. C, or G: B is C, G, or T; R is A or G; Y is C or T; and S is G or C.
  • TABLEs 1 and 2 provide LSRs and related RRS-1 and RRS-2 sites.
  • more than one RRS sequence may be provided for a given I., SR
  • the provided RRS sequences refer to RRS sequences of varying length, each of which comprises a core RRS sequence.
  • the RRS-2 sequence for LSR 2955954 can be SEQ ID NO: 3934, 9719, or 9792. wherein each of SEQ ID NOs: 3934 and 9792, comprise the core sequence of SEQ ID NO: 9719.
  • TABLE 5 provides exemplary ammo acid alterations relative to SEQ ID NO: 9401 useful in compositions, systems, and methods described herein.
  • TABLE 6 provides exemplary' amino acid alterations relative to SEQ ID NO: 9278 useful in compositions, systems, and methods described herein.
  • TABLE 7 provides exemplary effector protein CasPhi.12 engineered variant sequences.
  • TABLE 8 provides exemplary RDDP sequences.
  • TABLE 9 provides exemplary engineered RDDP sequences.
  • LSRs were identified from metagenomic data based on matches to the Recombinase and Resolvase Pfam domain HMMs. Additional LSRs were identified that matched the Recombinase Pfam and at least one other Pfam of interest. Open source software GeNomad was used to identify phage boundaries on the metagenomic contigs containing these LSRs. Shared palindromic sequences (from phage and genome itself) that occur around the phage boundaries and LSR boundaries were used to construct predicted attachment sites for each LSR. These attachment sites are iteratively shortened (in silico) on both ends to predict the “minimal attachment site” with the highest palindromi cits'.
  • LSR 005 Hie predicted genomic safe harbor for LSR 004, LSR 007 and LSR_012 is the albumin gene, and the predicted genomic safe harbor for LSR_005 is the AAVS1 gene.
  • Plasmid 1 (“pint”) contained the LSR enzyme coding sequence constitutively expressed under the CMC promoter, and plasmid 2 (“pAtt”) contained the putative respective attachment sequences, attB (RRS-2) and attP (RRS-1), flanking an EFS promoter sequence in reverse orientation with respect to an mCherry coding sequence.
  • the attB (RRS-2) and attP (RRS-1 ) sequences on the pAit plasmid are in opposing orientations surrounding the reverse EFS promoter such that only upon successful LSR-mediated recombination will the EFS promoter be” flipped” and in the correct orientation to drive mCherry expression.
  • the assay read-out to measure LSR activity is relative mCheny expression in the plnt+pAtt transfection condition relative to the pAtt-only transfection condition.
  • Controls The positive control for the assay was the well -characterized LSR Bxbl, and the negative control for each novel LSR was the p Att plasmid only condition to measure non- EFS promoter-driven background mCheny expression.
  • the positive hit criteria from the activity assay was set at 5% mCherry positive cells over-all, and 1 ,5x % mCherry positive cells in the plnt+pAtt condition relative to the pAtt-only control condition. Data is provided in FIG. 7.
  • the sequences of the LSRs that were tested are shown in TABLE 12 LSR 004 (SEQ ID NO: 16); LSR 005 (SEQ ID NO: 20); LSR 007 (SEQ ID NO: 3); and LSR 012 (SEQ ID NO: 18). All four tested LSRs demonstrated activity in mammalian cells.
  • LSR protein information and corresponding attB (RRS-2) and attP (RRS- 1) site containing sequences tested are provided in TABLE 13. The sequences of the LSRs that were tested are shown in TABLE 12.
  • Plasmid 1 contained the LSR enzyme coding sequence constitutively expressed under the CMV promoter
  • Plasmid 2 contained the putative respective attachment sequences, attB (RRS-2) and attP (RRS-1), flanking an EPS promoter sequence in reverse orientation with respect to an mCherry coding sequence.
  • the attB (RRS-2) and attP (RRS-1 ), sequences on the pAtt plasmid are in opposing orientations surrounding the reverse EFS promoter such that only upon successful LSR- mediated recombination will the EFS promoter be ⁇ 'Hipped” and in the correct orientation to drive mCherry expression
  • the assay read-out to measure LSR activity is relative mCheriy expression in the plnt+pAtt transfection condition relative to the pAtt-only transfection condition.
  • the positive control for the assay was the well-characterized LSR Bxbl, and the negative control for each novel LSR was the pAtt plasmid only condition to measure non-EFS promoter-driven background mCherry expression.
  • Plasmid 1 (“pint”’) contained the LSR enzyme coding sequence constitutively expressed under the CMV promoter, and plasmid 2 (‘"pAtt”) contained the putative respective attachment sequences, attB (RRS-2) and atP (RRS-1), flanking an mCheny sequence in reverse orientation with respect to an EFS promoter.
  • the attB (RRS-2) and attP (RRS-1) sequences on the pAtt plasmid are in opposing orientations surrounding the mCheny coding sequence such that only upon successful LSR- mediated recombination will the reporter be “flipped” and in the correct orientation to drive mCheny expression.
  • the assay read-out to measure LSR activity is relative mCheny expression in the plnt+pAtt transfection condition relative to the pAtt-only transfection condition.
  • the positive control for the assay was the well -characterized LSR Bxbl, and the negative control for each novel LSR was the pAtt plasmid only condition to measure non-EFS promoter-driven background mCheny expression. See FIG. 9 for a diagram of the assay.
  • results from the promoter swap assay are provided in FIG. 10 and results from the reporter swap assay are shown in FIG. 11. Both assays show that the tested LSRs are capable of provi ding recombination.
  • LSR_509/LSR_232, LSR. _512, LSR. 514, LSR_515, LSR_625, LSR_662, LSR_699/LSR_300, and LSR_828 were also identified as active in the reporter swap assay, and the data is shown in FIG. 13A-FIG. 13D. Swap Assay Hits Example 4. Integration of Large Payloads
  • Plasmids carrying DNA payloads as well as plasmids that encoded LSRs were cotransfected into HEK293T cells following the protocol disclosed in Example 3.
  • Payload plasmids contained the predicted recognition sequences for each of the tested LSRs.
  • LSR protein information is shown in TABLE 14.
  • the Bxbl recombinase was used as a control. Chromosomal integrations were detected using a modified GUEDE-seq assay. Results are shown in FIG. 12.
  • the black line shows the number of reads and the gray line shows the number of unique loci with the payload integrated.
  • Hie statistics reported in the figure reflect the sum of events across 5kb and 1 Okb payloads for each LSR. As shown in FIG. 12 there is a correlative relationship between the number of reads for each sequence that was integration and number of unique loci that included the integrated sequence.
  • TABLE 14 Tested LSRs
  • AttB and AttP sites were assessed for their ability to mediate integration of an mCherry reporter into the genome of HEK293 cells. Briefly , 300 ng plasmids (LSR plasmid and donor plasmids comprising an AttB or AttP site)) were lipofected into 30k HEK293T cells.
  • the cells were passaged every 3-4 days and analyzed via flow cytometry to see when the plasmids w ere removed.
  • Bxbl was used as a control, because of its low levels of integration at human pseudosites.
  • the negative control was a donor plasmid that contained a CasPhi sequence.
  • Three technical replicates were from the same DNA preparation. See overview in Fig. 14A. A complementary' AttB and AttP site are required for

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Abstract

Sont divulguées une composition et des méthodes de modification d'une molécule d'ADN double brin cible (ADNdb) dans une cellule. Des systèmes, des compositions et des méthodes peuvent comprendre une recombinase (par exemple, une grande recombinase sérine (LSR)), une protéine associée à CRISPR (Cas), une ADN polymérase ARN-dépendante, et/ou un ou plusieurs acides nucléiques guides, ou leurs utilisations. <i />
PCT/US2024/030106 2023-05-19 2024-05-17 Grandes recombinases de sérine et méthodes d'utilisation Pending WO2024243090A2 (fr)

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