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WO2024044723A1 - Rétrons modifiés et méthodes d'utilisation - Google Patents

Rétrons modifiés et méthodes d'utilisation Download PDF

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WO2024044723A1
WO2024044723A1 PCT/US2023/072872 US2023072872W WO2024044723A1 WO 2024044723 A1 WO2024044723 A1 WO 2024044723A1 US 2023072872 W US2023072872 W US 2023072872W WO 2024044723 A1 WO2024044723 A1 WO 2024044723A1
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Prior art keywords
retron
ncrna
gene editing
sequence
editing system
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WO2024044723A8 (fr
Inventor
Alim Ladha
Vladimir PRESNYAK
Inna SCHERBAKOVA
Brian Goodman
Mario Rodriguez MESTRE
Kyong-Rim KIEFFER-KWON
Socheata LY
Devin Scott QUINLAN
Muthusamy Jayaraman
Stephen Scully
Jacob LAYER
Abril Fleitas BEITANS
Zhongxia YI
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Renagade Therapeutics Management Inc
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Renagade Therapeutics Management Inc
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Priority claimed from US18/087,673 external-priority patent/US11866728B2/en
Priority claimed from PCT/US2023/061038 external-priority patent/WO2023141602A2/fr
Application filed by Renagade Therapeutics Management Inc filed Critical Renagade Therapeutics Management Inc
Priority to AU2023329392A priority Critical patent/AU2023329392A1/en
Priority to TW112132199A priority patent/TW202426060A/zh
Publication of WO2024044723A1 publication Critical patent/WO2024044723A1/fr
Publication of WO2024044723A8 publication Critical patent/WO2024044723A8/fr
Anticipated expiration legal-status Critical
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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    • 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]
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Provisional Application Serial No.63/488,317 (RTX007-P1), filed March 3, 2023, U.S. Provisional Application Serial No.63/491,603, filed March 22, 2023 (RTX008-P1), and U.S. Provisional Application Serial No.63/515,783, filed July 26, 2023 (RTX009-P1), each of which are incorporated herein by reference in their entireties.
  • This application references the following applications: U.S. Provisional Application Serial No.63/301,936, filed January 21, 2022 (RTX001-P1), and U.S. Provisional Application Serial No.63/370,880, filed August 9, 2022 (RTX002-P1), each of which are incorporated herein by reference in their entireties.
  • RNA-guided nucleases e.g., CRISPR nucleases
  • ZFN zinc-finger nucleases
  • TALENS transcription activator-like effector nucleases
  • HDR-dependent precise genome editing is the delivery of donor DNA template to the nuclease-induced DSB (e.g., see Ling et al., “Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates,” Science Advances, 2020, Vol.6, No.15, pp.1-8).
  • Various methods aimed at boosting the efficiency of HDR-dependent editing have been reported, many of which involve the physical tethering of the DNA donor to a component of the precise editing system. Exemplary methods have been discussed in: K.
  • Retrons are defined by their unique ability to produce an unusual satellite DNA known as msDNA (multicopy single-stranded DNA).
  • DNA encoding retrons includes a reverse trancriptase (RT)-coding gene (ret) and a nucleic acid sequence encoding the non-coding RNA (ncRNA), which contains two contiguous and inverted non-coding sequences referred to as the msr and msd.
  • RT reverse trancriptase
  • ncRNA nucleic acid sequence encoding the non-coding RNA
  • the ret gene and the non-coding RNA are transcribed as a single RNA transcript, which becomes folded into a specific secondary structure following post-transcriptional processing.
  • the RT binds the RNA template downstream from the msd locus, initiating reverse transcription of the RNA towards its 5 ⁇ end, assisted by the 2’OH group present in a conserved branching guanosine residue that acts as a primer.
  • Reverse transcription halts before reaching the msr locus, and the resulting DNA, the msDNA, remains covalently attached to the RNA template via a 2 ⁇ -5 ⁇ phosphodiester bond and base-pairing between the 3 ⁇ ends of the msDNA and the RNA template.
  • the external regions, at the 5 ⁇ and 3 ⁇ ends of the msd/msr transcript (a1 and a2, respectively) are complementary and can hybridize, leaving the structures located in the msr and msd regions in internal positions (see FIG.1A).
  • the msr locus which is not reverse transcribed, forms one to three short stem-loops of variable size, ranging from 3 to 10 base pairs, whereas the msd locus folds into a single/double long hairpin with a highly variable long stem of 10-50 bp in length that is also present in the final msDNA form.
  • retrons may be utilized as a means to provide donor DNA template for HDR-dependent genome editing (e.g., see Lopez et al., “Precise genome editing across kingdoms of life using retron-derived DNA,” Nature Chemical Biology, December 12, 2021, 18, pages199–206 (2022)), however, producing sufficient levels of donor DNA template intracellularly to sufficiently support efficient HDR-dependent editing remains a significant challenge.
  • Improved retron-based genome modification systems are highly desirous in the art.
  • the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron.
  • Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non-contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase.
  • the retron that becomes modifed with the one or more genetic modifications is a naturally occurring retron or retron component (e.g., naturally occuring ncRNA of Table A or RT) ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template.
  • a naturally occurring retron or retron component e.g., naturally occuring ncRNA of Table A or RT
  • HDR homology-dependent recombination
  • the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron-related sequence provided by Table X (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see Examples).
  • the recombinant retrons are based on introducing the one or more genetic modifications into previously available retron sequences (e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.
  • retron sequences e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA
  • the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT).
  • the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems).
  • recombinant retron components e.g., recombinant ncRNA and/or recombinant RT
  • programmable nucleases e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs,
  • the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems.
  • the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions.
  • the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles.
  • the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., ncRNA and mRNA delivered by LNPs
  • protein e.g., virus-like particles
  • RNP ribonucleoprotein
  • each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • an all-RNA system e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit.
  • a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit.
  • the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems.
  • the disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems.
  • this disclosure or the inventions herein provide a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo; the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the programmable nuclease; and each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c); whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c).
  • the delivery vehicle(s) comprise RNA cargo
  • the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered
  • (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retron reverse transcriptase.
  • (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.
  • the fusion protein comprises the C-terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the retron reverse transcriptase (nuclease:RT fusion); or the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (RT:nuclease fusion).
  • (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retron reverse transcriptase.
  • (c) is separate from (a)(i), (a)(ii) and (b) or is provided in trans.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 5’ end of the retron ncRNA.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 3’ end of the retron ncRNA.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the engineered ncRNA comprises a first guide RNA fused to the 5’ end of the retron ncRNA, and a second guide RNA fused to the 3’ end of the retron ncRNA, and the first and second guide RNAs target different sequences.
  • guide RNA for the programmable nuclease can comprise one or more guides that target the same or different target sequences.
  • Such guide RNA(s) in an embodiment can be single guide RNA(s) or sgRNA(s); for instance, when the nucleic acid programmable nuclease comprises a Cas9.
  • the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retron reverse transcriptase, and (b) the engineered retron ncRNA are in separate delivery vehicles.
  • the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle.
  • the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.
  • the engineered retron ncRNA includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm that hybridizes to a sequence 5’ to the target sequence and a 3' homology arm that hybridizes to a sequence 3' to the target sequence.
  • the donor polynucleotide can be heterologous to the cell.
  • the donor polynucleotide can be endogenous to the cell; for instance, the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state).
  • Such can be done in an animal cell, or a mammalian cell (e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse) or a human cell; for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human.
  • a mammalian cell e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse
  • a human cell for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human.
  • plant cells to introduce mutations that give rise to favorable phenotypic characteristics such as disease resistance or other favorable plant trait(s).
  • the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease.
  • the engineered retron ncRNA comprises: A) a pre-msr sequence having a first complementary region of the retron ncRNA; B) an msr sequence including an msr stem-loop structure; C) an msd sequence including an msd stem-loop structure and a sequence of interest, wherein said msd sequence templates a single strand DNA product (RT-DNA) in the presence of the retron reverse transcriptase; and D) a post- msd sequence having a second complementary region, wherein the first and second complementary regions form an a1/a2 duplex region of the retron ncRNA, wherein the msr stem- loop structure, the msd stem-loop structure, or the a1/a2 duplex comprise a modification which result in increased editing efficiency in the presence of a nucleic acid programmable nuclease that associates with the one or more guide RNAs, and wherein optionally one
  • the engineered retron ncRNA comprises A), B), C) and D
  • the sequence of interest can encode a donor polynucleotide comprising an intended edit to be integrated at a target sequence of a cell, wherein the donor polynucleotide is flanked by a 5' homology arm that hybridizes to a sequence 5’ to the target sequence and a 3' homology arm that hybridizes to a sequence 3' to the target sequence.
  • the engineered retron ncRNA comprises A), B), C) and D) (either with the sequence of interest encoding a donor polynucleotide or simply being a sequence of interest)
  • the ncRNA has a nucleotide sequence of Table B, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table B.
  • the donor polynucleotide can be heterologous to a cell. Alternatively, the donor polynucleotide can be endogenous to the cell.
  • the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state).
  • the gene editing system can comprise any combination(s) of the foregoing embodiments of the gene editing system.
  • this disclosure or the inventions herein provide a cell, such as an isolated cell comprising the gene editing system disclosed herein, such as in any of the foregoing paragraphs.
  • the cell e.g., isolated cell
  • the cell can be a eukaryotic cell.
  • the eukaryotic cell can be a plant cell or an animal cell or a mammalian cell, e.g., an isolated plant cell or an isolated animal cell or an isolated mammalian cell.
  • the mammalian cell e.g., an isolated mammalian cell
  • the cell can be a prokaryotic cell, e.g., a bacterial cell.
  • the donor polynucleotide can code for antibiotic susceptibility; and thus, the invention can involve a means for addressing antibiotic resistant bacteria by rendering such bacteria susceptible to antibiotics (and a subject to whom the gene editing system is administered can also then receive antibiotics to which the bacteria are rendered susceptible by the gene editing system).
  • this disclosure or the inventions herein provide a composition comprising: a) the gene editing system disclosed herein, such as in any of the foregoing paragraphs; and b)a pharmaceutically or veterinarily acceptable carrier.
  • the delivery vehicle in the composition can comprise a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d)one or more phospholipids.
  • the one or more ionizable lipids comprises an ionizable lipid set forth in Table 2.
  • this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in modifying a cell or genetically modifying a cell, e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell).
  • a cell or genetically modifying a cell e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell
  • this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in treating or addressing a genetic condition of a subject, [0033]
  • this disclosure or the inventions herein provide methods of genetically modifying a cell comprising: contacting a gene editing system as herein discussed, such as in any of the foregoing paragraphs, or a composition as herein discussed, such as in any of the foregoing paragraphs (which comprises a gene editing system as herein discussed, such as in any of the foregoing paragraphs), advantageously a gene editing system that includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, said method comprising contacting the composition or the gene editing system with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide
  • the cell can be a eukaryotic or a prokaryotic cell or an animal cell or a mammalian cell or a human cell or a bacterial cell or a plant cell.
  • a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo, said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the nucleic acid programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c). 2.
  • the delivery vehicle(s) comprise RNA cargo
  • said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA
  • the engineered retron ncRNA comprises an HDR nucleotide sequence substituted into a retron ncRNA; wherein the retron reverse transcriptase has an amino acid sequence comprising at least 90% sequence identity to a retron reverse transcriptase of Table A; wherein the retron ncRNA has about 85% to 98% sequence identity to a retron ncRNA of Table B. 3.
  • the retron ncRNA and the retron reverse transcriptase are from the same clade. 4.
  • the gene editing system of paragraph 2 wherein the retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to SEQ ID NO:15327, and the retron reverse transcriptase has at least 90% sequence identity to a type I-C retron reverse transcriptase. 5.
  • the retron reverse transcriptase comprises an amino acid sequence at least about 90% identical to SEQ ID NO:1262.
  • the retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to SEQ ID NO:16411, and the retron reverse transcriptase has at least 90% sequence identity to a type III retron reverse transcriptase. 7.
  • the retron reverse transcriptase comprises an amino acid sequence at least about 90% identical to SEQ ID NO:2781.
  • the retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to SEQ ID NO:18731. and the retron reverse transcriptase has at least 90% sequence identity to a type XIII retron reverse transcriptase.
  • the retron reverse transcriptase comprises an amino acid sequence at least about 90% identical to SEQ ID NO:6342.
  • the retron reverse transcriptase comprises at least one amino acid substitution that increases processivity and/or fidelity. 11.
  • the retron reverse transcriptase comprises an amino acid substitution in an amino acid residue that corresponds to the following amino acid residues in Eco1 RT: Q190, E302, or T306. 12. The gene editing system of paragraph 10, wherein the retron reverse transcriptase comprises an amino acid substitution in an amino acid residue that corresponds to the following amino acid substitutions in Eco1 RT: Q190F, E302R, or T306K. 13.
  • a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo, said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c), and wherein the engineered retron reverse transcriptase comprises a processivity enhancing domain or a fidelity enhancing domain.
  • the gene editing system of paragraph 13 wherein the processivity enhancing domain comprises Sso7d or Sac7d. 15. The gene editing system of paragraph 13, wherein the fidelity enhancing domain comprises a 3’ to 5’ exonuclease domain. 16. The gene editing system of paragraph 15, wherein the exonuclease domain comprises POLE1 POLD1, POLG, Pfu, or KOD. 17.
  • the engineered retron ncRNA comprises an HDR nucleotide sequence substituted into a retron ncRNA; wherein the retron reverse transcriptase has an amino acid sequence comprising at least 90% sequence identity to a retron reverse transcriptase of Table A; wherein the retron ncRNA has about 85% to 98% sequence identity to a retron ncRNA of Table B. 18.
  • the retron ncRNA and the retron reverse transcriptase are from the same clade. 19.
  • a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo, said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c), and wherein the engineered reverse transcriptase comprises a Y region domain that is from a retron RT that corresponds to the engineered retron ncRNA.
  • the fusion protein comprises the C- terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the retron reverse transcriptase (nuclease:RT fusion).
  • the fusion protein comprises the N- terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (RT:nuclease fusion). 25.
  • the engineered ncRNA comprises a first guide RNA fused to the 5’ end of the retron ncRNA, and a second guide RNA fused to the 3’ end of the retron ncRNA, and the first and second guide RNAs target different sequences.
  • the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP).
  • nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle.
  • nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.
  • the engineered retron ncRNA includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm that hybridizes to a sequence 5’ to the target sequence and a 3' homology arm that hybridizes to a sequence 3' to the target sequence.
  • the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease. 38.
  • nucleic acid programmable nuclease comprises a Cas9 nuclease.
  • nucleic acid programmable nuclease comprises a Cas9 nickase.
  • An isolated cell comprising the gene editing system of paragraph 1. 41. The isolated cell of paragraph 40, wherein the isolated cell is a mammalian cell. 42. The isolated cell of paragraph 41, wherein the mammalian cell is a human cell. 43.
  • a composition comprising: a) the gene editing system of paragraph 1; and b) a pharmaceutically or veterinarily acceptable carrier. 44.
  • composition of paragraph 43 wherein the delivery vehicle is a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.
  • the one or more ionizable lipids comprises an ionizable lipid set forth in Table 2. 46.
  • a method of genetically modifying a cell comprising: contacting the gene editing system of paragraph 1 with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein said guide RNA directs the complex to the target sequence, the nucleic acid programmable nuclease creates a double-stranded break in in the target sequence, the retron reverse transcriptase and engineered retron ncRNA create RT DNA that comprises the donor polynucleotide, and the donor polynucleotide becomes integrated at the target sequence, whereby editing the cell is genetically modified.
  • An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), said first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of a multi-copy single- stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus, wherein the ncRNA comprises: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%
  • the engineered nucleic acid construct of paragraph 2, wherein the second polynucleotide comprises: III) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; and/or IV) encodes a consensus amino acid sequence of Table C, or encodes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
  • An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), said first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of a multi-copy single- stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT or portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the m
  • An engineered nucleic acid construct comprising: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein said msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct optionally has (a) a secondary structure of a wild-type ncRNA of any one of FIGs.2-27 or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettere
  • the engineered nucleic acid construct of any one of paragraphs 1 to 4a comprising one or more sequence modifications (e.g., an insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: a) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); b) modulates (e.g., reduces) immunogenicity of ncRNA encoded by the engineered retron (e.g., the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); c) modulates (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or d) modulates (e.g., improves) efficiency of targeted genome editing /
  • the one or more heterologous nucleic acid sequences comprise: a) a heterologous nucleic acid (such as the coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as in an S region (e.g., S1, S2, S3 and/or S4), or the tip of an L region (e.g., L1, L2, L3 and/or L4), or upstream or downstream of either the msr locus or the msd locus; or b) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted either upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a guide RNA.
  • a heterologous nucleic acid such as the coding sequence for an RNA aptamer or a ribozyme
  • said protein or peptide of interest comprises a therapeutic protein useful in treating a disease.
  • a sequence-specific nuclease such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)
  • a sequence-specific nuclease such as a CRISPR/Cas effector enzyme, a ZFN,
  • the sequence-specific nuclease is fused to the RT, optionally via a flexible linker (e.g., a flexible linker comprising Gly and Ser rich sequences such as G4S repeats or GS repeats) or by a generally disordered protein sequence (such as unstructured hydrophilic, biodegradable protein polymer, e.g., an XTEN peptide polymer).
  • a flexible linker e.g., a flexible linker comprising Gly and Ser rich sequences such as G4S repeats or GS repeats
  • a generally disordered protein sequence such as unstructured hydrophilic, biodegradable protein polymer, e.g., an XTEN peptide polymer.
  • nuclease is a CRISPR/Cas effector enzyme that forms a complex with a guide RNA (gRNA) recognizing a target sequence, wherein the gRNA is linked to the ncRNA and/or the msDNA, either directly or through a linker / spacer polynucleotide.
  • gRNA guide RNA
  • the DNA-repair modulating biomolecule is a regulatory protein that modulates (e.g., enhances) HDR, and the regulatory protein is fused to the RT or to the sequence-specific nuclease, optionally via a flexible linker (e.g., the flexible linker comprising Gly and Ser rich sequences such as G4S repeats or GS repeats) or by a generally disordered protein sequence (such as unstructured hydrophilic, biodegradable protein polymer, e.g., an XTEN peptide polymer). 17.
  • a flexible linker e.g., the flexible linker comprising Gly and Ser rich sequences such as G4S repeats or GS repeats
  • a generally disordered protein sequence such as unstructured hydrophilic, biodegradable protein polymer, e.g., an XTEN peptide polymer.
  • a vector system comprising one or more vectors comprising the engineered nucleic acid construct of any one of paragraphs 1-16, wherein the vector system is optionally all- RNA.
  • the vector system of paragraph 17 wherein the msr locus, the msd locus, and the polynucleotide encoding the RT are comprised within the same vector.
  • the same vector further comprises a promoter operably linked to the msr locus and/or the msd locus.
  • the promoter is further operably linked to the polynucleotide encoding the RT. 21.
  • a vector system comprising one or more vectors, comprising the engineered nucleic acid construct of paragraph 1 or 2, wherein the vector system further comprises a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the msr locus, the msd locus, and the second polynucleotide encoding the RT are provided by at least two different vectors. 22.
  • RT reverse transcriptase
  • the second polynucleotide comprises: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; and/or b) the second polynucleotide encodes: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at
  • non-viral vector comprises a plasmid.
  • non-viral vector comprises a liposome, a lipid nanoparticle (LNP), a cationic polymer, a vesicle, or a gold nanoparticle.
  • LNP lipid nanoparticle
  • cationic polymer a cationic polymer
  • a vesicle a vesicle
  • gold nanoparticle a gold nanoparticle.
  • sequence-specific nuclease comprises an RNA-guided sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme, an engineered RNA-guided FokI-nuclease (e.g., dCas-FokI), an RNA-guided DNA endonuclease, TnpB, IscB, or a transposon-associated nuclease), or a non-RNA-guided sequence-specific nuclease (e.g., a meganuclease, a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), or a restriction endonuclease (RE)).
  • RNA-guided sequence-specific nuclease e.g., a CRISPR/Cas effector enzyme, an engineered RNA-guided FokI-nuclease (e.g., dCas-F
  • the Cas effector enzyme is a Class 1, Type I, II, or III Cas; a Class 2, Type II Cas (e.g., Cas9); or a Class 2, Type V Cas (e.g., Cpfl). 32.
  • the RNA-guided sequence-specific nuclease comprises the CRISPR/Cas effector enzyme, the engineered RNA-guided FokI-nuclease (e.g., dCas-FokI), the RNA- guided DNA endonuclease, TnpB, IscB, IsrB, or the transposon-associated nuclease; or, 2) non-RNA-guided sequence-specific nuclease comprises the meganuclease, the zinc finger nuclease (ZFN), the TALE nuclease (TALEN), or the restriction endonuclease (RE).
  • ZFN zinc finger nuclease
  • TALEN TALE nuclease
  • RE restriction endonuclease
  • An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single- stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a sequence encoding a reverse transcriptase (RT), or a domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%
  • An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single- stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; wherein the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,
  • An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single- stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; wherein, the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%
  • An isolated host cell comprising the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any of paragraphs 17-33, the RNA molecule of paragraph 34, or the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37.
  • 39. The isolated host cell of paragraph 38, wherein the host cell is a prokaryotic, archeon, or eukaryotic host cell.
  • 40. The isolated host cell of paragraph 38, wherein the eukaryotic host cell is a mammalian host cell. 41.
  • the isolated host cell of paragraph 39, wherein the eukaryotic host cell is a non-human host cell.
  • 42. The isolated host cell of paragraph 40 wherein the mammalian host cell is a human host cell. 43.
  • a pharmaceutical composition comprising: a) the engineered nucleic acid construct of any one of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any one of paragraphs 17-33, the RNA molecule of paragraph 34, the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, and/or the isolated host cell of any one of paragraphs 38-43; and b) a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising: a) a lipid nanoparticle (LNP); and b) the engineered nucleic acid construct of any one of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any one of paragraphs 17-33, the RNA molecule of paragraph 34, and/or the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37.
  • lipid nanoparticle comprises: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.
  • the one or more ionizable lipids is selected from the group consisting of those disclosed in Table 2.
  • the pharmaceutical composition of paragraph 47 or 48 wherein the one or more structural lipids are selected from the group consisting of cholesterol, fecosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, and hydrocortisone. 50.
  • the pharmaceutical composition of any one of paragraphs 47-49, wherein the one or more PEGylated lipids are selected from the group consisting of PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.
  • any one of paragraphs 47-50, wherein the one or more phospholipids are selected from the group consisting of 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-oc
  • lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol% of PEG lipid.
  • the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol% of PEG lipid.
  • the LNP further comprises a targeting moiety operably connected to the LNP.
  • the LNP further comprises one or more additional components selected from the group consisting of DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200.
  • the lipid nanoparticle comprises at least one cationic lipid selected from the group consisting of: a lipid in Table 2, a lipid having a structure of Formula (I), a lipid having a structure of Formula (II), a lipid having a structure of Formula (III), a lipid having a structure of Formula (IV), a lipid having a structure of Formula (V), a lipid having a structure of Formula (VI), and combinations thereof.
  • a lipid in Table 2 a lipid having a structure of Formula (I), a lipid having a structure of Formula (II), a lipid having a structure of Formula (III), a lipid having a structure of Formula (IV), a lipid having a structure of Formula (V), a lipid having a structure of Formula (VI), and combinations thereof.
  • a kit comprising the engineered nucleic acid construct of any one of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any one of paragraphs 17-33, the RNA molecule of paragraph 34, the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, the host cell of any one of paragraphs 38-43, or the pharmaceutical composition of any one of paragraphs 44-56, and instructions for genetically modifying a cell with said engineered nucleic acid construct, ncRNA, vector system, host cell, or pharmaceutical composition. 58.
  • a method of modifying a target DNA sequence in a host (e.g., mammalian) cell comprising introducing into the mammalian cell the engineered nucleic acid construct of any one of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any one of paragraphs 17- 33, the RNA molecule of paragraph 34, the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, or the pharmaceutical composition of any one of paragraphs 44-56, to allow production of the msDNA in the host (e.g., mammalian) cell, wherein the heterologous nucleic acid in the msDNA is integrated into the genome of the host (e.g., mammalian) cell at the target DNA sequence by homology-dependent recombination.
  • a method of treating a disease or condition in a subject in need thereof comprising administering a therapeutically effective amount of the engineered nucleic acid construct of any one of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid construct of any one of paragraphs 1-16, the vector system of any one of paragraphs 17-33, the RNA molecule of paragraph 34, the engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, the host cell of any one of paragraphs 38-43, or the pharmaceutical composition of any one of paragraphs 44-56 to the subject, thereby treating the disease or condition in the subject.
  • a method of treating a disease or condition in a subject in need thereof comprising administering a therapeutically effective amount of the host cell of any one of paragraphs 38-43 to the subject, thereby treating the disease or condition in the subject.
  • the host cell is autologous to the subject.
  • the host cell is allogeneic to the subject.
  • FIG.1A is a schematic that depicts naturally occurring retrons from genomic DNA stage through production of the msDNA chimeric microsatellite molecule.
  • Retrons are encoded in the bacterial genome and comprise a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT).
  • ncRNA and RT initially are transcribed from the retron DNA as a single polycistronic message.
  • the initial transcript is processed resulting in the removal or separation of the transcript encoding the retron RT.
  • the remaining transcript is the ncRNA, which undergoes folding to form a secondary structure having several characteristic stem-loops and a duplex formed between the 5 ⁇ and 3 ⁇ regions of the ncRNA (i.e., the a1/a2 duplex).
  • the folded ncRNA is recognized by the accompanying RT which is separately translated and provided in trans.
  • the translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region.
  • the RT initiates reverse transcription of the RNA towards its 5 ⁇ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA.
  • G conserved guanosine
  • FIG.1B is a schematic depicting an embodiment of a recombinant retron contemplated by this disclosure.
  • a nucleic acid molecule comprises a nucleotide sequence encoding a retron ncRNA region (the msr/msd region) is depicted at the top left.
  • the msr region has been modified by introducing one or more nucleotide modifications (e.g., a nucleotide substitution, deletion, or insertion).
  • nucleotide modifications e.g., a nucleotide substitution, deletion, or insertion
  • the modified msr is referred to as msr’.
  • the msd has been modified by introducing a heterologous nucleotide sequence encoding an HDR donor template.
  • the retron DNA has been modifed to introduce a nucleotide sequence encoding a guide RNA at the 3 ⁇ end of the retron DNA sequence.
  • a DNA vector e.g., a plasmid.
  • the DNA is shown to be transcribed as a polycistronic message that includes the msr’/msd’ region (forming the ncRNA) which is fused at its 3 ⁇ end to the guide RNA (in other embodiments, the guide RNA could be fused to the 5 ⁇ end of the retron ncRNA.
  • This intermediate is shown to form a complex with a reverse transcriptase provided in trans (e.g., by way of a separate expression vector or delivered mRNA).
  • the top right schematic shows the formation of a complex between the recombinant ncRNA and the RT and the beginning of reverse transcription from the covalently-linked conserved guanosine (G) (i.e., the “priming G” or “priming guanosine”) using the msd RNA as a template sequence.
  • G covalently-linked conserved guanosine
  • FIG.1C is a schematic depicting a recombinant retron-based genome editing system described herein.
  • the components of such a system may include (a) a guide RNA provided in cis (e.g., fused to the recombinant retron msDNA) and/or in trans (e.g., separately expressed in a cell), (b) a recombinant ncRNA (including at least a sequence encoding an HDR donor template and optionally a guide RNA fused ot the ncRNA), (c) a reverse transcriptase, and (d) a programmable nuclease.
  • a guide RNA provided in cis (e.g., fused to the recombinant retron msDNA) and/or in trans (e.g., separately expressed in a cell)
  • a recombinant ncRNA including at least a sequence encoding an HDR donor template and optionally a guide RNA fused ot the ncRNA
  • a reverse transcriptase e.g., a reverse transcriptas
  • FIG.1D provides a simplified schematic of a the natural lifecycle of a retron.
  • Retrons typically comprise a reverse transcriptase (RT) and two non-coding contiguous inverted sequences (msr and msd) transcribed as a single RNA that is folded into a specific secondary structure.
  • RT reverse transcriptase
  • msr and msd non-coding contiguous inverted sequences
  • the conserved NAXXH motif and VTG triplet in retron RTs are indicated.
  • the RT binds downstream from the msd locus in the RNA, initiating reverse transcription of the RNA template towards its 5 ⁇ end, assisted by the 2 ⁇ OH group present in a conserved branching G residue acting as a primer.
  • FIG.1E provides a detailed representation of the natural biological pathway of a retron in a cell, concluding with the generation of the msDNA satellite molecule. This figure parallels FIG.1D but more completely depicts the stages of msDNA production.
  • (1) depicts the retron locus which includes an ncRNA locus having an msr locus and a msd locus (both of which are non-coding) and a reverse transcriptase (RT) locus.
  • the ncDNA locus and the RT locus are transcribed as a single RNA transcript, which is depicted in (2).
  • the colors representing each component in (1) are carried through each of stages (2) through (6).
  • Stages (3) and (4) depict the folding of the ncRNA portion into a series of stem loops, wherein the 5 ⁇ end and the 3 ⁇ ends of the ncRNA form a duplex.
  • the position of the conserved branching guanosine residue having a 2’OH group is show.
  • the branching guanosine serves as a future priming site for the reverse transcriptase.
  • Stage (4) further shows that the region of the transcript encoding the reverse transcriptase is removed, separately being translated to produce the reverse transcriptas enzyme.
  • the reverse transcriptase associates with the folded ncRNA and begins polymerization of a single strand of DNA (i.e., the reverse transcription product) from the primer site (i.e., the conserved branching guanosine residue having the 2’OH end) and using the msd RNA sequence as a template. Reverse transcription terminates at the msr region.
  • the msd RNA template is exonucleotically removed, thereby resulting in a chimeric molecule comprising the msr RNA region which is covalently joined to the ssDNA transcription product through covalent linkage to the conserved guanosine primer residue.
  • FIG.1F is a schematic depicting that the herein disclosed recombinant retron- based genome modification systems may be implemented as (a) cell recorder systems, (b) genome editing systems, and (c) recombineering systems. These uses are not intended to be limiting.
  • FIG.1G is a schematic depicting various configurations contemplated for the recombinant retron disclosed herein.
  • FIG.1H is a schematic that emphasizes that any suitable configuration for presenting the components of the recombinant retron-based genome modification systems disclosed herein to a cell are contemplated, including where the RT and/or the programmable nuclease are provided in trans relative to the retron ncRNA.
  • the RT and the programmable nuclease may be provided as a fusion protein.
  • FIG.1I depicts that the RT and programmable nuclease may be provided as fusion proteins (top, middle) or provided separate from one another.
  • FIG.1J depicts that nuclear localization signals may be engineered into the polypeptides of the disclosure (e.g., a RNA-guide nuclease) to facilitate translocation into the nuclease of cell where editing occurs.
  • FIG.1K is a schematic (not to scale) representation depicting an embodiment of genome editing, in which double-stranded break (DSB) created by a suitable nuclease (such as a CRISPR/Cas effector enzyme, a ZFN, TALEN, meganuclease, TnpB, IscB, or restriction enzymes (Res)) promotes the insertion of a donor or template sequence (shown here as a “marker” flanked by homologous sequences matching those flanking the DSB, provided on a “donor vector”).
  • DSB double-stranded break
  • a suitable nuclease such as a CRISPR/Cas effector enzyme, a ZFN, TALEN, meganuclease, TnpB, IscB, or restriction enzymes (Res) promotes the insertion of a donor or template sequence (shown here as a “marker” flanked by homologous sequences matching those flanking the
  • FIG.1L depicts the capability of three different kinds of retrons (Eco1-R1, Eco3– R2, Eco5-R3) to insert a 16 base pair insertion at a specific genomic site, the EMX1 gene.
  • FIG.1M outlines a procedure used to evaluate the capability of a retron to produce genomic insertions.
  • FIG.2 (SEQ ID NO:19970) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IA/IIA1 retron produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.3 (SEQ ID NO:19971) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IB1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.4 (SEQ ID NO:19972) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IB2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.5 (SEQ ID NO:19973) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IC retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.6 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA other retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.7 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.8 (SEQ ID NO:19976) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA3 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.9 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA4 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.10 (SEQ ID NO:19978) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA5 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.11 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.12 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.13 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of IIIA3 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.14 (SEQ ID NO:19982) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA4 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.15 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA5 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.16 (SEQ ID NO:19984) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIunk retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.17 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IV retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.18 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IX retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.19 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type V retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.20 (SEQ ID NO:19988) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type VI retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.21 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XI Group 1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.22 (SEQ ID NO:19990) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XI Group 2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.23 (SEQ ID NO:19991) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XII retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.24 (SEQ ID NO:19992) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XIII retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.25 (SEQ ID NO:19993) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XIV retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.26 (SEQ ID NO:19994) is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Ec107 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved).
  • Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.27 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Outgroup A retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.
  • the colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.
  • FIG.28 is a phylogenetic tree of RT sequences constructed in accordance with Example 3.
  • FIG.29 is a structural representation of the retron loci associated with each retron type in FIG.28.
  • FIG.30 shows the position of certain retrons (EcoI, Eco3, Eco5, AcoI, RTX003_2042, RTX003_6083v1, and RTX003_6943) within the phylogenetic retron tree of FIG.28.
  • FIG.31A is a plasmid map of an exemplary retron (EcoI) tested in the Examples herein.
  • FIG.31B is a linear representation in 5’ to 3’ direction of the plasmid map of FIG.31A.
  • FIG.31C is a plasmid map of an exemplary retron (RTX3_6083v1) tested in the Examples herein.
  • FIG.31D is a linear representation in 5’ to 3’ direction of the plasmid map of FIG.31C.
  • FIG.32 is a representation of a plasmid-based assay used to measure retron precise edits and indels as performed in the Examples.
  • Step 1 a plasmid (e.g., that of FIG. 31A or FIG.31C) is transfected into human cells (e.g., HEK293t cells) which are engineered to express Cas9. Editing is allowed to occur for 72 hours at 37°C.
  • Step 2 the genomic DNA is extracted from the cells and used to prepare a next-generation sequencing (NGS) library for sequencing.
  • the library is sequenced over the target site (e.g., EMX1) of editing to generate sequence reads.
  • Step 3 the sequencing reads are analyzed to obtain a frequency of sequence reads containing the desired edit (percentage of precision editing) and a frequence of indels at the desired edit site (percentage of indels).
  • FIG.33 is an equivalent representation of FIG.32.
  • FIG.34 is a representation of a methodology for transfecting HEK293T cells. Cells were seeded in 24 well plate one day prior to transfection.
  • FIG.35 (SEQ ID NOs:19996-20003) (Top to Bottom) is an example of reference sequence and desired editing outcome for Eco3 retron at he EMX1 genomic site. Analysis of editing outcomes is performed using CRISPresso2 pipeline.
  • FIG.36 shows the results of plasmid-based assay (e.g., according to FIG.33) demonstrating up to about 0.3% precise edits and as low as 40% indels with Eco1 retron in Cas9 expressing HEK293T cells.
  • the plasmid that encodes Eco1 RT and Eco1 ncRNA-sgRNA fusion targeting EMX1 was transfected via lipofection using two different amounts of Lipofectamine.
  • FIG.37 shows the results of plasmid-based assay (e.g., according to FIG.33) demonstrating up to about 0.1% precise edits and as low as 3% indels with AcoI.
  • Aco1 retron has not been experimentally validated to produce msDNA. Precise editing activity observed in this experiment strongly support that Aco1 retron is capable of generating msDNA inside human cells.
  • FIG.38 shows the results of plasmid-based assay (e.g., according to FIG.33) demonstrating up to about 0.3% precise edits and as low as 5% indels with RTX003_2042. This retron can achieve a comparable precise editing to Eco1 but with significantly lower indels (10- fold).
  • RTX003_2042 is a novel retron and precise editing activity observed in this experiment strongly support that RTX003_2042 retron could generate msDNA inside human cells.
  • FIG.39A shows the results of plasmid-based assay demonstrating up to about 0.05 ⁇ 0.08% precise edits and as low as 2.5 ⁇ 4% indels with RTX003_6083v1 and 6943. Both are novel retrons and precise editing activity observed in this experiment strongly support that RTX003_6083v1 and 6943 retron could generate msDNA inside human cells.
  • FIG.39B shows follow up experiments using the same assay of FIG.39A indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 fold more precise edits than Eco1 while indels generated from these two retrons were 2-3 fold lower.
  • RTX3_2042 showed precise editing at similar frequencies to Eco1 but had more variability than other samples.
  • FIG.39C shows follow up experiments using the same assay of FIG.39A indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 fold more precise edits than Eco1 while indels generated from these two retrons were 2-3 fold lower.
  • RTX3_2042 showed precise editing at similar frequencies to Eco1 but had more variability than other samples.
  • FIG.39D shows the results of plasmid-based assay demonstrating up to about 0.7% precise edits and as low as ⁇ 4% indels with RTX003_0637, RTX003_1262, and RTX003_6342 compared to empty vector and EcoI.
  • RTX003_0637, RTX003_1262, and RTX003_6342 are novel retrons and precise editing activity observed in this experiment strongly support that these retrons could generate msDNA inside cells.
  • FIG.39E shows the results of plasmid-based assay demonstrating precise editing and indel generation for an array of retrons, including EcoI, Eco3, RTX3_2042_RT_inactivated, RTX3_2042, RTX3_6083v1, RTX3_6943, RTX3_6943, RTX3_1262, RTX3_6342S, and RTX3_6342L.
  • FIG.40 is a representation of an two-RNA editing assay used in the Examples to measure the relative editing efficiency of exemplary retrons using electroporation-based delivery of two RNA components into HEK293T cells.
  • FIG.41 shows the results of two-RNA system (RT mRNA + ncRNA-sgRNA fusion) delivered to Cas9 expressing HEK293T cells by electroporation. Eco1, Eco3 and Eco5 retrons were tested. Results showed precise edits (left graph) up to 0.4% for Eco3 and as low as 10% indels (right graph) for Eco3.
  • FIG.42 shows the results of titration of two-component Eco3 RNA system (RT mRNA + ncRNA-sgRNA fusion) delivered to Cas9 expressing 293T cells by electroporation.
  • the RT mRNA and the ncRNA were mixed at ratios of 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, respectively, and delivered at two different amounts of RT mRNA (0.2 or 0.5 ⁇ g).
  • FIG.43 is a representation of a three-RNA retron editing system which involves delivery by electroporation of three RNA components (RT mRNA, retron ncRNA-sgRNA fusion, and Cas9 mRNA) into HEK293T cells.
  • FIG.44 shows the results of Cas9 mRNA titration of three-component Eco3 RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA) delivered to 293T cells by electroporation.
  • FIG.45 depicts a process of lipofection using three RNA system in HEK293T cells. Cells were seeded in 96 well plate one day prior to transfection.
  • FIG.46 shows the results of three-component Aco1 RNA system (RT mRNA + ncRNA +Cas9 mRNA) delivered to HEK293T cells by lipofection.
  • FIG.47 shows the results of minimal Cas9 nuclease activity when sgRNA is fused to ncRNA of Eco3 retron.
  • FIG.48 is a representation of the all-RNA editing assay used in the Examples to measure the relative editing efficiency of the sample retrons in an all-RNA format, modified with a step of in trans guide RNA spike-in. Electroporation using three RNA system + sgRNA trans spike-in in HEK293T cells.
  • FIG.49 shows the results of guide RNA spike-in in all RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA + sgRNA) delivered to HEK293T cells by electroporation.
  • the guide RNA spike-in in all RNA system increased precise editing up to ⁇ 50 fold.
  • the increasing amount of guide RNA gradually increased precise editing and 1:8 of RT mRNA:ncRNA-sgRNA fusion performed slightly better than 1:6, reaching 13% of precise editing.
  • frequency of indels is shown for respective conditions.
  • FIG.50 represents a lipofection process using three RNA system + gRNA trans spike-in in HEK293T cells.
  • Cells were seeded in 96 well plate one day prior to transfection. Appropriate amount of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA, sgRNA and Lipofectamine reagent were mixed and transferred to cells. After 72 hours incubation, genomic DNA was extracted and targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.
  • FIG.51 shows the results of guide RNA spike-in (i.e., delivering a separate molecule bolus of guide RNA which in all RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA + sgRNA) delivered to HEK293T cells by lipofection.
  • RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA + sgRNA RNA spike-in
  • the amount of guide RNA spike- in is titrated at 2, 5 and 10 ng.
  • the guide RNA spike-in in all RNA system increased precise editing up to 3.5 fold, 12% of efficiency. The increasing amount of guide RNA at this range did not further increase precise editing.
  • FIG.52 shows the results of ncRNA-sgRNA fusion separation in all RNA system (RT mRNA + Cas9 mRNA + either ncRNA-sgRNA fusion OR separate ncRNA + sgRNA) delivered to HEK293T cells by lipofection.
  • RT mRNA + Cas9 mRNA + either ncRNA-sgRNA fusion OR separate ncRNA + sgRNA delivered to HEK293T cells by lipofection.
  • the amount of guide RNA spike-in is titrated at 0, 2, 5, 10, 50 and 100 ng.
  • precise editing peaked at 2.23% compared to 1.78% with ncRNA-sgRNA fusion.
  • FIG.53 is a schematic of improved templates used for in vitro transcription to produce ncRNA (left or A) and ncRNA modifications (right or B).
  • A relates to the optimization of RNA production by in vitro transcription.
  • Previously made in vitro transcription experiments to produce RNA used a double-stranded DNA template containing a 3’ overhang (on same strand as T7 promoter sequence).
  • a new template with a blunt end was designed and tested and as shown in FIG.54 results in increased precise editing efficiency.
  • FIG.54 shows the results of ncRNA-sgRNA fusion separation in 4 component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount as shown on the graphs. Using RNA generated from linearized plasmid template containing a 3’ overhang produced 1.35% precise edits.
  • FIG.55 (SEQ ID NO: 19969) provides schematics for end protection of RNA from cellular nuclease activity by capping and tailing.
  • A a 7-methylguanosine cap0 was added to 5’ triphosphate of RNA.
  • B a poly-A tail was added to the 3’ end by enzymatic addition. Tail length is estimated over 50 nucleotides.
  • FIG.56 shows the result of end protection of ncRNA-sgRNA fusion by cap and tail in 4 component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA-sgRNA 400ng, Cas9 mRNA 100 ng, and sgRNA 5 ng.
  • RT mRNA + Cas9 mRNA + ncRNA + sgRNA 4 component all-RNA system
  • ncRNA-gRNA fusion was either capped (+cap –tail) or poly-A tailed (-cap +tail) or both capped and poly-A tailed (+cap +tail).
  • RNA without end protection (-cap –tail) produced ⁇ 4.5 % precise edits and the editing was dependent on retron since the absence of RT abrogated precise editing.
  • RNA with either or both protection by cap and tail produced lower precise editing (left graph) but lowered indels (right graph) than without cap and tail.
  • FIG.57 shows the result that shortening msd stems can modulate precising editing in a retron specific manner.
  • Modified retrons include RTX3_4536 (long (L) and short (S) versions), RTX3_6279 (long (L) and short (S) versions), RTX3_6342 (long (L) and short (S) versions), RTX3_6438 (long (L) and short (S) versions), RTX3_6549 (long (L) and short (S) versions), and RTX3_6605 (long (L) and short (S) versions).
  • FIG.58 shows the result that shortening msd stems can modulate precising editing in a retron specific manner.
  • Modified retrons include RTX3_5752 (long (L) and short (S) versions), RTX3_6221 (long (L) and short (S) versions), and RTX3_6034 (long (L) and short (S) versions).
  • FIG.59A Demonstrates that four retrons from various clades are capable of insertions of up to 100 bps to EMX1 locus.
  • Figure shows the results of testing different template lengths for four different retrons in all RNA system delivered to HEK293T cells by lipofection. At given amount of RT mRNA and Cas9 mRNA, ncRNA-sgRNA fusion with different template lengths ranging from 10 to 100 bp were added.
  • FIG.59B Demonstrates that four retrons from various clades are capable of insertions of up to 100 bps to EMX1 locus.
  • Figure shows indel results corresponding with the constructs of FIG.59A, graphing frequency of indels for respective conditions.
  • FIG.60A Demonstrates that additional sgRNA targeting the same genomic locus increases the indel and insert frequencies.
  • FIG.60B Demonstrates that additional sgRNA targeting the same genomic locus increases the indel and insert frequencies. This figure reports the frequency of indels associated with the retrons of FIG.60A.
  • FIG.61A is a schematic showing unedited and edited allele by GFP gene insertion at EMX1 locus using retron editing system.
  • FIG.61B Demonstrates three retrons from various clades are capable of GFP integration at EMX1 locus.
  • the figure provides qPCR data depicting GFP insertion at 5’ or 3’ junctions at EMX1 locus by three retrons.
  • ncRNA-sgRNA engineered to contain GFP gene insert was transfected to HEK293T cells, together with RT mRNA of each retron, Cas9 mRNA, with/without additional sgRNA. Genomic DNA was analyzed by indicated primer pairs to detect GFP gene insertion at 5’ or 3’ junctions on the edited allele.
  • ⁇ Ct was obtained by subtracting +RT sample’s Ct value from –RT sample’s Ct value for relative quantification of GFP insertion at EMX1 locus.
  • the bigger value of ⁇ Ct indicates the higher frequency of insert in the sample.
  • the figure provides qPCR data depicting GFP insertion at 5’ or 3’ junctions at EMX1 locus by two retrons.
  • ncRNA-sgRNA engineered to contain GFP gene insert was transfected to HEK293T cells, together with RT mRNA of each retron, Cas9 mRNA, with/without additional sgRNA.
  • Genomic DNA was analyzed by indicated primer pairs in the table to detect GFP gene insertion at 5’ or 3’ junctions on the edited allele.
  • ⁇ Ct was obtained by subtracting junction Ct value from EMX1 Ct value for relative quantification of GFP insertion at EMX1 locus. With all two retrons, amplicons of both 5’ and 3’ junctions are detectable significantly above background level.
  • FIG.61D shows PCR products for Aco1 were run on Agilent Tapestation High Sensitivity D1000 screentape. In the presence of RT (+RT sample, left on the gel), the expected size of amplicons were detected at both 5’ and 3’ junctions of edited alleles as indicated by arrows. Edited alleles were not detected in the absence of RT (-RT sample, right on the gel). Triangles indicate the expected amplicon for unedited alleles, which is present in both -RT and +RT samples.
  • FIG.62A Demonstrates that order of sgRNA and ncRNA within fused RNA wherein sgRNA is positioned upstream of ncRNA is more active in precise editing and indels.
  • frequency of indels is shown for respective conditions.
  • FIG.62B Demonstrates that where sgRNA is positioned downstream of ncRNA, additional sgRNA compensates for lower editing activity of configuration. Same as previous figure but with the addition of sgRNA, In the presence of additional of sgRNA, the differences between ncRNA-sgRNA and sgRNA-ncRNA depends on each retron. For Eco3 and R2042, sgRNA-ncRNA performed better than ncRNA-sgRNA. For Aco1, ncRNA-sgRNA performed slightly better than sgRNA-ncRNA. On the bottom row of graphs, frequency of indels is shown for respective conditions. [00150] FIG.63 Demonstrates that RT and Cas9 are required for precise editing.
  • ncRNA/ncRNA-sgRNA/sgRNA-ncRNA were added. All insertion templates tested were 25 bp. The best ncRNA format depends on each retron. For Eco3, the highest precise editing was observed with separate ncRNA + additional sgRNA (9.88%). For Aco1, the highest precise editing was observed with ncRNA-sgRNA + additional sgRNA (2.96%). For R2042, the highest precise editing was observed with separate ncRNA + additional sgRNA (7.4%). For R6943, the highest precise editing was observed with ncRNA-sgRNA fusion + additional sgRNA (0.39%).
  • FIG.65 Demonstrates that retrons can achieve precise editing at AAVS1 gene locus. Results of testing four different retrons to insert 25 bp template into AAVS1 locus. Retrons were delivered as all RNA to HEK293T cells by lipofection. For all four retrons, the best precise editing was observed in the presence of additional sgRNA and ranged from 0.73% to 2.05% depending on the retron used. On the right, frequency of indels is shown for respective conditions. [00153] FIG.66 Demonstrates that retrons can achieve precise editing at AAVS1 gene locus. Results of negative controls for AAVS1 experiments in previous figure.
  • FIG.67 Demonstrates that template for either target or non-target strand can be used for precise editing. Results of testing templates on different strands relative to Cas9 sgRNA target strand for two different retrons in all RNA system delivered to HEK293T cells by lipofection. At given amount of RT mRNA, Cas9 mRNA and additional sgRNA, ncRNA- sgRNA was added. All insertion templates tested were 25 bp.
  • Cas9 nickase activity in 4 component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA/ncRNA-sgRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA/ncRNA-sgRNA 400ng, Cas9 variant mRNA 100 ng, and sgRNA 5 ng. Either separated ncRNA or ncRNA-sgRNA fusion was used. Precise editing by Cas9 WT (Wild Type) was ⁇ 9% when either ncRNA or ncRNA-sgRNA was used with additional sgRNA.
  • FIG.69 Demonstrates that retron mediated precise editing by Cas9 nickase is dependent on each component of all RNA system.
  • FIG.70 demonstrates that dual sgRNAs increases precise editing, but not indels. The figure shows the results of testing dual sgRNAs for R2042 in all RNA system delivered to HEK293T cells by lipofection.
  • FIG.71 Precise editing and indel results in HEK293T cells administered by lipofection an all-RNA retron editing system based on R6083 in various configurations. Configurations included testing different template lengths (25 bp – 100 bp) with fused ncRNA- sgRNA constructs (with and with spiked sgRNA) and separated ncRNA and sgRNA constructs. At given amount of RT mRNA and Cas9 mRNA, ncRNA-sgRNA fusion with different template lengths ranging from 25 to 100 bp or ncRNA with 25bp insertion were added. Precise insertion of 100 bp was observed at ⁇ 2% efficiency.
  • FIG.72 Results of testing R6083 retron to insert 25 bp template into AAVS1 locus in HEK293T cells. Retron (ncRNA-sgRNA fusion), retron RT, and Cas9 components were delivered as all RNA to HEK293T cells by lipofection. With additional sgRNA, 3% of editing efficiency was observed and the activity was completely dependent on the presence of either RT or ncRNA.
  • FIG.73 Demonstrates that the separation of Cas9 and RT enzymes shows higher editing efficiency than when they are fused in retron editing with Eco3.
  • the same molar concentration of Cas9 or RT mRNA as in Cas9-RT fusion format were used in separation and the editing efficiency of fused or separated enzyme were compared for either Eco3 ncRNA (left on left graph) or Eco3 ncRNA-sgRNA fusion (right on right graph).
  • FIG.74 Demonstrates that both cap and tail modifications to ncRNA significantly increased editing efficiency about 2-fold. Results of end protection of ncRNA-sgRNA fusion by cap and tail in 4 component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX.
  • ncRNA-gRNA fusion was either capped (+cap –tail) or poly-A tailed (-cap +tail) or both capped and poly-A tailed (+cap +tail).
  • RNA without end protection (-cap –tail) produced ⁇ 4.5 % precise edits and the editing was dependent on retron since the absence of RT abrogated precise editing. While capping alone did not change editing efficiency, tailing alone slightly increased editing and both cap and tail significantly increased editing efficiency about 2-fold (left graph). Indels at all conditions were comparable in right graph.
  • FIG.75 Schematics of ncRNA circularization which as demonstrated in FIG.76 resulted in increase editing efficiency.
  • Step 1. ncRNA is flanked by internal homology, intron and another homology sequences outward toward both ends.
  • Step 2. Two homology sequences facilitate closer proximity of both ends.
  • Exogenous GTP initiates a series of trans-esterification
  • Step 3. introns are excised and ncRNA is ligated to a circular form.
  • FIG.76 Results of testing modified ncRNAs (3’MS2 modification and circularized ncRNA) for precise editing in 4 component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA 400ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. With the addition of MS2 stem loop at 3’ end of ncRNA, the activity nearly doubled at 15% efficiency from unmodified ncRNA ( ⁇ 8%). Circularization of ncRNA achieved further increase of the efficiency, reaching editing efficiency of 22%.
  • FIG.77 Results suggest pairing between RT and its cognate ncRNA are required for precise editing. Results of pairing RT and ncRNA from same or different retrons for precise editing at EMX1 locus in HEK293T cells by all-RNA system. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA 400ng, Cas9 mRNA 100 ng, and sgRNA 5 ng.
  • FIG.78 Retron editing supports insertion of exon-sized long insertions of up to 305 bp. Results of testing exon-size long insertiona at EMX1 locus with Aco1, Eco3 and R1262 retrons. Aco1 achieved precise insertions of 10, 100 and 205bp at 8, 4, and 1.3% (left graph).
  • FIG.79 Demonstrates that novel retron R1262 inserts 205 bp insertion at AAVS1 at over 11% efficiency. Results of testing 205bp insertion at AAVS1 locus with a novel retron R1262. R1262 obtained 25 and 205bp insertion at 25%, and 11.1 % efficiency respectively. As controsl, precise editing was dependent on the presence of RT and sgRNA.
  • FIG.80 Optimization of ratios of RT to ncRNA for precise editing by R1262 to install 205-305 bp insertions.
  • FIG.81 NHEJ-based insertion of retron-made double strand DNA at target site A. Some retrons could produce double strand DNA using msR-msD hybrid as a primer (e.g. Sen1). Desired sequence to insert in the genome flanked by guide-RNA recognition sequence is integrated in msD of ncRNA.
  • ncRNA is designed as in A to contain a sequence to insert at the target site flanked with guide-RNA recognition sequences.
  • Second ncRNA includes a reverse complementary sequence of the first ncRNA in order that two of them form a duplex.
  • Such double strand DNA is trimmed by nuclease-guide RNA complex and inserted to the target site cleaved by nuclease-guide RNA complex.
  • FIG.82A Non-coding RNA optimization A. There are number of structural components in retron ncRNA, 1.
  • FIG.82B Non-coding RNA optimization B. Separation of msR RT primer and msD RT template allows chemical modification of msR RT primer. In combination with end protection of msD RT template by cap and tail, the stability of ncRNA could be enhanced. Use of reverse complementary sequences at 3’ end of RT primer and 5’ end of msD RT template (depicted in dashed lines) stabilizes the primer/template complex.
  • FIG.83A provides a schematic outline a modified retron gene editing system that comprises an engineered variant reverse transcriptase (RT) with improved fidelity and/or processivity.
  • the engineered RT is produced by introducing selective amino acid substitutions into the sequence of a retron RT at residue positions that are orthologous to those altered residues in Murine Leukemia Virus (MMLV) RT that result in improved fidelity and processivity of MMLV RT.
  • Step (a) a structural alignment is constructed as between a retron RT and an engineered MMLV RT having one or more amino acid substitutions that are associated with improved fidelity and processivity in the MMLV RT context.
  • a retron gene editing system may comprise in (d) a ncRNA, a guide, a programmable nuclease (or an RNA encoding same), and the engineered RT (or an RNA encoding same).
  • Step (e) the components (which can be in all RNA format) are delivered to a cell (e.g., by LNP delivery), which results in (f) the targeted single (in the case of a nickase) or double strand cut, followed by (g) targeted repair by the reverse transcription product of the ncRNA at the cut site. This results in (h) DNA with a precise edit.
  • FIG.83B provides a summary of mutations for installing in RTX3_1262 RT and RTX3_6242, as well as in Eco1 RT that are orthologous to known beneficial amino acid substitutions made in MMLV that are reported in the literature.
  • FIG.83C provides a structural alignment between RTX3_6342 RT (yellow) and MMLV-RT (Protein Data Bank 4MH8) (green), which reveals that the N-terminus of the RTX3- 6342 RT may be able to be truncated, for example, at the suggested points of trunctation.
  • FIG.83D shows three dimensional structures of MMLV RT proximal to Q190 and the corresponding orthogonol positions Q161 in Eco1. In the top pictures, the glutamine is conserved in both MMLV and Eco1.
  • FIG.83E Mutations in MMLV RT and Eco1 that improve processivity. Provides three dimensional structures of E302 of MMLV RT and the corresponding reside of G283 in Eco1, both of which appear to be involved in binding to nucleic acid. Residues in nucleic acid binding domains can be mutated to enforce stronger interactions.
  • MMLV E302R creates a positive charge to interact with the negatively charged template.
  • Eco1 G283 is in a conserved DNA binding alpha helix. Mutating G283 and orthologous retron residues to a positively charged amino acid may improve processivity.
  • FIG.83F Mutations in MMLV RT and Eco1 that improve processivity. Provides three dimensional structures of T306 of MMLV RT and the corresponding reside of F287 in Eco1, both of which appear to be involved in binding to nucleic acid. Residues in nucleic acid binding domains can be mutated to enforce stronger interactions.
  • MMLV T306K creates a positive charge to interact with the negatively charged template.
  • FIG.84 describes the making of variant retron RTs by saturation mutagenesis of targeted domains involved in processivity and fidelity (e.g., palm, fingers, and thumb domains).
  • An engineered RT is produced by introducing saturation mutagenesis into palm, finger, and thumb domains in a retron RT that are identified through a structural alignment with MMLV RT.
  • Step (a) a structural alignment is constructed as between a retron RT and an engineered MMLV RT having one or more amino acid substitutions that are associated with improved fidelity and processivity in the MMLV RT context, particularly in the palm, finger, and thumb domains.
  • Step (b) the palm, finger, and thumb domains are identified.
  • Step (c) the palm, finger, and/or thumb domains are mutagenized using a saturation mutagenis methodology and the resultant mutants are screened in an activity assay to select one or more leads variants that have increased processivity and/or fidelity.
  • Step (e) the components (which can be in all RNA format), including the lead retron RT variants, are delivered to a cell (e.g., by LNP delivery), which results in (f) the targeted single (in the case of a nickase) or double strand cut, followed by (g) targeted repair by the reverse transcription product of the ncRNA at the cut site. This results in (h) DNA with a precise edit.
  • FIG.85A describes the making of variant retron RTs by constructing chimeric RT proteins that fuse a “Y region” of a retron RT (i.e., the region that is reported to be responsible for binding to the msr region of a retron ncRNA) with another RT (e.g, an MMLV RT) or replacing the “Y region” or one retron RT with that of another retron RT.
  • a retron RT i.e., the region that is reported to be responsible for binding to the msr region of a retron ncRNA
  • another RT e.g, an MMLV RT
  • the chimeric protein could be engineered against any particular ncRNA to have the corresponding Y region that would be expected to bind to that ncRNA.
  • FIG.85B (SEQ ID NOS:19955-19968) (Top to Bottom) provides a multiple sequence alignment of amino acid sequences of numerous retron RTs (top).
  • the boxed region indicates the position of a conserved VTG triplet that marks the N-terminal side of the Y region (as reported in Anna J Simon, Andrew D Ellington, Ilya J Finkelstein, Retrons and their applications in genome engineering, Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Pages 11007–11019, which is incorporated herein by reference), which is about 90 amino acids in length.
  • the schematic below is a magnified version of the top figure.
  • FIG.86 describes the making of variant retron RTs by fusing one or more processivity-enhancing factors, such as Sso7d and Sac7d, and/or one more fidelty-enhancing factors, e.g., 3’>5’ exonuclease domains to increase proofreading activity (e.g., POLE1, POLD1, POLG, Pfu, or KOD).
  • processivity-enhancing factors such as Sso7d and Sac7d
  • fidelty-enhancing factors e.g., 3’>5’ exonuclease domains to increase proofreading activity (e.g., POLE1, POLD1, POLG, Pfu, or KOD).
  • FIG.87 Electroporation using plasmid in K562 cells.
  • FIG.88 Results of plasmid-based assay in K562 cells demonstrating up to about 1 ⁇ 10% precise edits and as low as 5 ⁇ 30% indels with RTX003_2042, 6083v1, 6943, 1262, 6342L and 6342S. All are novel retrons and precise editing activity observed in this experiment strongly support that these novel retrons could generate msDNA inside K562 cells.
  • FIG.89 Results of plasmid-based assay comparing literature annotated retrons to novel retrons in K562 cells. These data demonstrate that novel retrons RTX3_1262, RTX3_6342L, and RTX3_6342S can generate about 15-19% precise edits and as low as 20 ⁇ 35% indels. Novel retrons perform as well or better than Aco1 and better than the other literature validated retrons: Eco1, Eco3, Sau1, and Sen1.
  • FIG.90 Results of plasmid-based assay comparing RTX3_2781 to lead retrons RTX3_1262 and RTX3 _6342S/L in K562 cells. These data demonstrate that RTX3_2781 installs precise edits at comparable efficiencies to RTX3_1262 and 6342S/L.
  • FIG.91 Results of plasmid-based assay in K562 cells demonstrating up to about 0.6% precise edits and as low as 5% indels with RTX003_2042, 6083v1, 6943, 1262, 6342L and 6342S using a Cas9 D10A nickases.
  • FIG.92 Results of plasmid-based assay in K562 cells demonstrating up to 0.5% precise edits using the LbCas12a nuclease. Precise editing in this system strongly suggests the generation of msDNA inside cells and compatibility with Cas12a-like nucleases for gene- editing.
  • FIG.93 Results of plasmid-based assay in K562 cells demonstrating up to 0.5% precise edits using the TnpB nuclease.
  • FIG.94 RTX3_6342 (predicted structure from alpha-fold)(yellow) aligned to MMLV-RT (PDB 4MH8) (green).
  • Native RTX3_6342 RT is fused to a non-RT related domain at the N-terminus that may not be necessary for reverse transcription. Arrows mark potential truncation points of interest. Jumper et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). https://doi.org/10.1038/s41586-021-03819-2.
  • FIG.95 Results of plasmid-based assay in K562 cells demonstrating up to about ⁇ 20% precise edits and ⁇ 20% indels with RTX003_6342S and truncation variants. Precise editing activity observed in this experiment strongly support that these truncations retain the capability to generate msDNA inside cells.
  • FIG.96 Results of plasmid-based assay in K562 cells demonstrating up to ⁇ 40% precise edits and ⁇ 60% indels with RTX003_6342S and RTX3_1262 with differing insert sizes.
  • FIG.97 Results of plasmid-based assay in K562 cells demonstrating up to ⁇ 40% precise edits and ⁇ 40% indels with RTX003_6342S and RTX3_1262 with differing insert sizes while ignoring or quantifying substitutions during CRISPResso2 analysis.
  • FIG.98 Results of plasmid-based assay in K562 cells screening mutations in RTX3_6342 reverse transcriptase predicted to interact with ncRNA after structural alignment of a predicted alphafold RTX3_6342 structure and a crystal structure of Retron Eco1 with ncRNA.
  • FIG.99 Results of plasmid-based assay in K562 cells demonstrating that orthologous MMLV mutations in RTX3_6342S do not consistently increase the installation frequencies of inserts >205bp.
  • FIG.100 Results of plasmid-based assay in K562 cells screening mutations in RTX3_6342 reverse transcriptase predicted to interact with ncRNA after structural alignment of a predicted alphafold RTX3_6342 structure and a crystal structure of Retron Eco1 with ncRNA.
  • the E238R, E479K and K255P are mutations that potentially increase the installation frequencies of 305 bp inserts at the EMX1 locus.
  • FIG.101 Predicted structures of RTX3_6342 wildtype and with different N- terminal truncations. Predicted structures were generated with Alphafold and aligned to the Eco1/CryoEM structure (79VU.pdb) using PyMoL align.
  • FIG.102 Results of plasmid-based assay in K562 cells assessing the effects of partial or full deletions of N-terminal helices of RTX3_6342 on the installation frequencies of longer inserts. We observed that partial N-terminal truncation boost the precise edits/indel ratio of longer inserts.
  • FIG.103 Results of plasmid-based assay in K562 cells assessing the effects of DNA-binding domain fusion to RTX3_6342 on the installation frequencies of 405 bp inserts.
  • FIG.104 Results of plasmid-based assay in K562 cells screening retron family members similar to RTX3_6342, 6083, 6943, or 1262. We noted that most retrons with robust gene-editing activity were in the RTX3_6342 family.
  • FIG.105 Results of testing precise editing activity of a novel reton R2781 in all- RNA system.
  • R2781 demonstrated 10 bp insertion at EMX1 locus in plasmid system, at similar activity as other hits, R1262, R6342S, 6342L.
  • the activity of R2781 for 25 bp, 205 bp and 405 bp insertion at EMX1 locus was evaluated with two different homology arm (HA) length (30 bp for both arms or 49 bp left/65 bp right). Precise insertion of 25 bp with 30 bp homology arm was achieved at 11% efficiency. The efficiency of same length insertion was reduced to half with longer homology arm (49/65 bp) suggesting that this retron reverse transcriptase might have limited enzymatic processivity.
  • HA homology arm
  • FIG.106 Results of testing precise editing activity of a novel retron R6342S in all-RNA system with Cas.9 D10A. Previously, it was demonstrated that retron R6342S could achieve precise editing with Cas9 WT. Here, the activity for 25 bp insertion in EMX1 locus was evaluated in 293T cells with either Cas9 D10A or Cas9 D10A R221K/N394K mutant and either 10 or 50 ng sgRNA.
  • FIG.107 Results of testing precise deletion activity of a novel reton R1262 in all- RNA system. In top panel, two deletion strategies are shown. Retron ncRNA is designed by juxtaposing left and right homology arms sequences to delete the intervening sequence.
  • Del1 is deleting 214 bp upstream of Cas9 cutting site and del2 is deleting 248bp downstream of Cas9 cutting site at EMX1 locus.
  • Retron mediated deletion was compared with direct delivery of increasing amount of single strand DNA (ssDNA) donor (150 and 300 ng).
  • Bottom panel, left graph shows that R1262 was able to delete 248bp (del2) from EMX1 locus at similar activity as ssDNA donor. Indels are shown at the right graph at respective conditions of left graph.
  • unintended indel activity by retron-mediated deletion was ⁇ 6 fold lower than ssDNA donor mediated deletion.
  • FIG.108 Results of testing non-homologous end joining (NHEJ) based insertion activity of a novel reton R1262 and R6342 in all-RNA system.
  • NHEJ non-homologous end joining
  • Retron reverse transcriptase generates complementary single strand DNAs from two ncRNAs that contain either sense or anti-sense insert sequences.2.
  • Complementary single strand DNAs form duplex 3.
  • Cas9-sgRNA complex remove Y-shape single stranded portion (msR and msD spacer sequences) at both extremes.4.
  • FIG.109 Results of testing immune response to retron RNAs.
  • Human peripheral blood mononuclear cells hPBMCs
  • PBMCs contain both innate and adaptive immune cells and they are equipped with sensors to detect foreign nucleic acids.
  • Frozen hPBMCs were thawed to rest for overnight.
  • RT mRNA with U or m1 ⁇ and ncRNA with/without cap0 (m7G) at 5’ end were electroporated individually or together as indicated in the label, along with unmodified GFP mRNA from TriLink as a control. After overnight culture, the supernatant was analyzed for cytokine production. Among 25 cytokines and chemokines examined, those detected above detection limit are shown.
  • FIG.110 Results of testing retron-mediated gene editing in human stem progenitor cells (HSPCs) in all-RNA system. Bone marrow derived CD34+ human stem projenitor cells (HSPCs) were used to evaluate the performance of retron-mediated gene editing in primary cells.
  • HSPCs human stem progenitor cells
  • HSPCs Frozen HSPCs were thawed to expand for three days in the presence of hSCF, hFLT3-L, hTPO cytokines to prevent differentiation.
  • Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA and ncRNA was mixed at mass ratio indicated in each label, and electroporated to HSPCs by Lonza electroporator using two different programs. After additional three-day culture, genomic DNA was extracted and sequenced to measure editing frequency. With total of 5 microgram of RNA, divided at 1: 0.3 : 4: 1 Cas9 :gRNA :ncRNA :RT, precise insertion of 25 bp at AAVS1 locus was observed at 0.1% of frequency (left graph).
  • FIG.111 Results of testing retron-mediated gene editing in human T-cells in all- RNA system.
  • Human pan T-cells from peripheral blood were used to evaluate the performance of retron-mediated gene editing in primary cells.
  • Frozen T-cells were thawed and activated for two days in the presence of anti-CD3/anti-CD28 conjugated magnetic beads and IL-2 cytokine.
  • Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA and ncRNA was mixed at mass ratio indicated in each label, and electroporated to T-cells by either Neon or Lonza electroporator. After additional three-day culture, genomic DNA was extracted and sequenced to measure editing frequency.
  • FIG.112 provides a schematic of retron ncRNA library. Variants of different elements in the ncRNA (modified a1:a2 stem, msR, msD regions) are associated with unique barcodes and the library is synthesized as an oligo library. By sequencing barcodes inserted at the genomic locus, the efficiency of associated ncRNA variants can be measured in human cells.
  • FIG.113 outlines the variant library of Example 31.
  • FIG.114 provides a schematic depicting the screening process used to screen the ncRNA variant library described in Example 31.
  • FIG.115 provides a scatter plot summarizing the results of FIG.31. In the scatter plot, each dot represents one variant with relative genomic insertion level on the y-axis and ssDNA production level on the x-axis. Dotted lines represent the levels of genomic insertion and ssDNA production from WT R6342S control. Hence, the ncRNA variants on the upper right part of the dotted lines are outperforming WT R6342S control on both levels.
  • biologically active refers to a characteristic of an agent (e.g., DNA, RNA, or protein) that has activity in a biological system (including in vitro and in vivo biological system), and particularly in a living organism, such as in a mammal, including human and non-human mammals.
  • agent e.g., DNA, RNA, or protein
  • an agent when administered to an organism has a biological effect on that organism, is considered to be biologically active.
  • Bulge refers to a small region of unpaired base(s) that interrupts a “stem” of base-paired nucleotides.
  • the bulge may comprise one or two single- stranded or unbase-paired nucleotides joined at both ends by base-paired nucleotides of the stem.
  • the bulge can be symmetrical (viz., the two unbase-paired single-stranded regions have the same number of nucleotides), or asymmetrical (viz., the unbase-paired single stranded region(s) have different or unequal numbers of nucleotides), or there is only one unbase-paired nucleotide on one strand.
  • a bulge can be described as A/B (such as a “2/2 bulge,” or a “1/0 bulge”) wherein A represents the number of unpaired nucleotides on the upstream strand of the stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem.
  • cDNA refers to a strand of DNA copied from an RNA template, e.g., by a reverse transcriptase.
  • Cognate refers to two biomolecules that normally interact or co-exist in nature.
  • nucleic acid e.g., RNA, DNA
  • RNA nucleic acid
  • anneal or “hybridize”
  • nucleic acid specifically binds to a complementary nucleic acid
  • Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA].
  • adenine (A) pairing with thymidine (T) adenine (A) pairing with uracil (U)
  • 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 base-pairing with codons in mRNA.
  • a guanine (G) is considered complementary to both a uracil (U) and to an adenine (A).
  • U uracil
  • A adenine
  • 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.
  • sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide 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, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically 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 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) 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.
  • DNA-guided nuclease is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.”
  • An example of a DNA-guided nuclease is reported in Varshney et al., DNA-guided genome editing using structure-guided endonucleases, Genome Biology, 2016, 17(1), 187, which may be used in the context of the present disclosure and is incorporated herein by reference.
  • DNA- guided nuclease or “DNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the DNA-guided nuclease.
  • the guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence.
  • the DNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing.
  • DNA regulatory sequences can be used interchangeably herein to 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 and/or regulate translation of a mRNA into an encoded polypeptide.
  • a non-coding sequence e.g., guide RNA
  • a coding sequence e.g., coding sequence and/or regulate translation of a mRNA into an encoded polypeptide.
  • Donor nucleic acid or “donor polynucleotide” or “donor DNA” or “HDR donor DNA” it is meant a single-stranded DNA to be inserted at a site cleaved by a programmable nuclease (e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN; a meganuclease) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like).
  • a programmable nuclease e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN; a meganuclease
  • the donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g.70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g., within about 200 bases or less of the target site, e.g., within about 190 bases or less of the target site, e.g., within about 180 bases or less of the target site, e.g., within about 170 bases or less of the target site, e.g., within about 160 bases or less of the target site, e.g., within about 150 bases or less of the target site, e.g., within about 140 bases or less of the target site, e.g., within about 130 bases or less of the target site, e.g., within about 120 bases or less of the target site, e.g., within about 110 bases or less of the target site, e.g., within about 100 bases or less of the target site, e.
  • RNA 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), a “non-coding” RNA (ncRNA), a guide RNA, etc.).
  • the retron DNA may encode the ncRNA loci (which includes the msr and msd regions) as well as a retron RT.
  • Engineered retron As used herein, the term “engineered retron” or equivalently, “recombinant retron,” refers to a retron that does not occur in nature.
  • engineered retrons can include wildtype or naturally-occurring retrons that are modified to contain at least one modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92
  • nucleotide of a starting point retron e.g., a wildtype retron
  • the nucleotides may be contiguous or non-contiguous.
  • an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature.
  • an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic / artificial / recombinant nucleic acid sequences.
  • an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences.
  • modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci.
  • Linking guide RNA molecules to the 5 ⁇ and/or 3 ⁇ ends also represent another modification envisioned by the recombinant retrons disclosed herein.
  • the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons.
  • Exosomes [00226] As used herein, the term “exosomes” refer to small membrane bound vesicles with an endocytic origin.
  • exosomes are generally released into an extracellular environment from host/progenitor cells post fusion of multivesicular bodies the cellular plasma membrane.
  • exosomes can include components of the progenitor membrane in addition to designed components (e.g. engineered retron).
  • Exosome membranes are generally lamellar, composed of a bilayer of lipids, with an aqueous inter-nanoparticle space.
  • Expression vector or “expression construct” refers to a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses.
  • Numerous vectors and expression systems are commercially available, such as from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the present invention comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof.
  • RNA molecules that binds to a programmable nuclease of a retron-based gene editing systems and which targets the nuclease to a specific location within the targeted polynucleotide sequence is referred to herein as the “guide RNA” or “guide RNA polynucleotide” (also referred to herein as a “guide RNA” or “gRNA” or “crRNA”).
  • a guide RNA comprises two segments, a “DNA-targeting segment” and a “protein-binding segment.”
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA.
  • a protein-binding segment of a guide RNA can comprise base pairs 5-20 of the RNA molecule that is 40 base pairs in length; and the DNA-targeting segment can comprise base pairs 21-40 of the RNA molecule that is 40 base pairs in length.
  • the definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and may include regions of RNA molecules that are of any total length and may or may not include regions with complementarity to other molecules.
  • the DNA-targeting segment (or “DNA-targeting sequence”) comprises a nucleotide sequence that is complementary to a specific sequence within a targeted polynucleotide sequence (the complementary strand of the targeted polynucleotide sequence) designated the “protospacer-like” sequence herein.
  • the protein-binding segment interacts with a site-directed modifying polypeptide.
  • site-directed modifying polypeptide is a CRISPR nuclease
  • site-specific cleavage of the targeted polynucleotide sequence may occur at locations determined by both (i) base-pairing complementarity between the guide RNA and the targeted polynucleotide sequence; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the targeted polynucleotide sequence.
  • PAM protospacer adjacent motif
  • heterologous nucleic acid sequence refers to a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (e.g., DNA or RNA) and, if expressed, can encode a heterologous polypeptide.
  • a cellular sequence e.g., a gene or portion thereof
  • the heterologous sequence inserted into the wild- type retron regions does not naturally insert into such regions (e.g., the engineered retron with the inserted heterologous sequence is not naturally existing).
  • the heterologous sequence can be from the same species of bacteria in which the wild-type retron is normally found, so long as the heterologous sequence is not naturally inserted in the wild-type retron at the location in which the heterologous sequence is inserted.
  • the heterologous sequence is a mammalian sequence (e.g., a human sequence), or a reverse complement thereof.
  • Heterologous nucleic acid sequences introduced into retrons can including without limitation guide RNA sequences, HDR donor templates, protein-encoding genes, or non- coding functional RNA elements (e.g., stem-loops, hairpins, and bulges).
  • Homology-directed repair refers to the specialized form DNA repair that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and leads to the transfer of genetic information from the donor to the target.
  • Homology-directed repair may result in an alteration of the sequence of the target molecule (e.g., insertion, deletion, mutation), if the donor polynucleotide differs from the target molecule and part or all of the sequence of the donor polynucleotide is incorporated into the targeted polynucleotide sequence.
  • Identical refers to two or more sequences or subsequences which are the same.
  • substantially identical refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a comparison algorithm or by manual alignment and visual inspection.
  • two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the “percent identity” of two or more sequences.
  • the identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence.
  • substantially identical or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.
  • Stringent hybridization conditions and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
  • LNP Lipid nanoparticle
  • the term “lipid nanoparticle” or LNP refers to a type of lipid particle delivery system formed of small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space.
  • LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.
  • LNPs may comprise a nucleic acid (e.g.
  • an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid.
  • an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid.
  • Linker refers to a molecule linking or joining two other molecules or moieties.
  • the linker can be an amino acid sequence in the case of a linker joining two fusion proteins.
  • an RNA-guided nuclease e.g., Cas12a
  • the linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together.
  • a ncRNA at its 5 ⁇ and/or 3 ⁇ ends may be linked by a nucleotide sequence linker to one or more guide RNAs.
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30- 35, 35-40, 40- 45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
  • Liposomes [00237] As used herein, the term “liposomes” refer to a type of lipid particle delivery system comprising small vesicles that contain at least one lipid membrane surrounding an aqueous inner-nanoparticle space that is generally not derived from a progenitor/host cell.
  • Liposomes are a versatile carrier platform in that they are capable of transporting hydrophobic or hydrophilic molecules, including small molecules, proteins, and nucleic acids into cells. They were the earliest developed generation of nanoscale medicine delivery platform. Numerous liposomal drug formulations have been approved for human medicines, e.g., Doxil, a lipid nanoparticle formulation of the antitumor agent doxorubicin. Further discuss of liposomes can be found, for example, in Tenchov et al., “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp. 16982-17015 (the contents of which are incorporated by reference).
  • loop in a polynucleotide refers to a single stranded stretch of one or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, wherein the most 5 ⁇ nucleotide and the most 3 ⁇ nucleotide of the loop are each linked to a base-paired nucleotide in a stem.
  • Micelles As used herein, the term “micelles” refer to small particles which do not have an aqueous intra-particle space.
  • Nanoparticle As used herein, the term “nanoparticle” refers to any nanoscale particle typically ranging in size from about 1 nm to 1000 nm.
  • nuclear localization sequence refers to an amino acid sequence that promotes import of a protein (e.g., a RNA-guided nuclease) into the cell nucleus, for example, by nuclear transport.
  • Nuclear localization sequences are known in the art. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences.
  • nucleic acid or “nucleic acid molecule” or “nucleic acid sequence” or “polynucleotide” generally refer to deoxyribonucleic or ribonucleic oligonucleotides in either single- or double-stranded form. The term may also encompass oligonucleotides containing known analogues of natural nucleotides.
  • RNA Ribonucleic acid
  • DNA DNA
  • cDNA including RT DNA
  • DNA or RNA
  • nucleotides Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) also may (or may not) encompass nucleotide modifications, e.g., methylated and/or hydroxylated nucleotides, e.g., Cytosine (C) encompasses 5-methylcytosine and 5- hydroxymethylcytosine.
  • nucleic acid-guided nuclease or “nucleic acid-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide nucleic acid (e.g., a guide RNA or a guide DNA) thereby forming a complex between the guide nucleic acid and the nucleic acid-guided nuclease.
  • the guide nucleic acid comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence.
  • the nucleic acid-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide nucleic acid, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing.
  • the nucleic acid- guided nuclease will include a DNA-binding activity (e.g., as in the case for CRISPR Cas9).
  • nucleic acid-guided nuclease is programmed by associating with a guide RNA molecule and in such cases the nuclease may be called “RNA-guided nuclease.” When programmed by a guide DNA, the nuclease may be called a “DNA-guided nuclease.”
  • Nucleic acid-guided, RNA-guided, or DNA-guided nucleases may also be referred to as “programmable nucleases,” which also include other classes of programmable nucleases which associate with specific DNA sequences through amino acid / nucleotide sequence recognition (e.g., zinc fingers nucleases (ZFN) and transcription activator like effector nucleases (TALEN)) rather than through guide RNAs.
  • ZFN zinc fingers nucleases
  • TALEN transcription activator like effector nucleases
  • any nuclease contemplated herein may also be engineered to remove, inactivate, or otherwise eliminate one or more nuclease activities (e.g., by introducing a nuclease-inactiving mutation in the active site(s) of a nuclease).
  • a nuclease that has been modified to remove, inactivate, or otherwise eliminate all nuclease activity may be referred to as a “dead” nuclease.
  • a dead nuclease is not able to cut either strand of a double-stranded DNA molecule.
  • a nuclease that has been modified to remove, inactivate, or otherwise eliminate at least one nuclease activity but which still retains at least one nuclease activity may be referred to as a “nickase” nuclease.
  • a nickase nuclease cuts one strand of a double-stranded DNA molecule, but not both strands.
  • a CRISPR Cas9 naturally comprises two distinct nuclease activity domains, namely, the HNH domain and the RuvC domain. The HNH domain cuts the strand of DNA bound to the guide RNA and the RuvC domain cuts the protospacer strand.
  • RNA-guided nuclease may be similarly converted to nickases and/or dead nucleases by inactivating one or more of the existing nuclease domains.
  • operably linked refers to the correct location and orientation in relation to a polynucleotide (e.g., a coding sequence) to control the initiation of transcription by RNA polymerase and expression of the coding sequence, such as one for the msr gene, msd gene, and/or the ret gene.
  • a polynucleotide e.g., a coding sequence
  • Other transcriptional control regulatory elements e.g., enhancer sequences, transcription factor binding sites
  • Programmable nuclease is meant to refer to a polypeptide that has the property of selective localization to a specific desired nucleotide sequence target in a nucleic acid molecule (e.g., to a specific gene target) due to one or more targeting functions.
  • targeting functions can include one or more DNA-binding domains, such as zinc finger domains characteristic of many different types of DNA binding proteins or TALE domains characteristic of TALEN proteins.
  • Such targeting function may also include the ability to associate and/or form a complex with a guide RNA, which then localizes to a specific site on the DNA which bears a sequence that is complementary to a portion of the guide RNA (i.e., the spacer of the guide RNA).
  • the programmable nuclease may be a single protein which comprises both a domain that binds directly (e.g., a ZF protein) or indirectly (e.g., an RNA-guided protein) to a target DNA site, as well as a nuclease domain.
  • the programmable nuclease may be a composite of two or more separate proteins or domains (from different proteins) which together provide the necessary functions of selective DNA binding and nuclease activity.
  • the programmable nuclease may comprise a (a) nuclease-inactive RNA-guided nuclease (which still is capable of binding a guide RNA, localizing to a target DNA, and binding to the target DNA, but not capable of cutting or nicking the strands) fused to a (b) nuclease protein or domain, such as a FokI nuclease.
  • promoter refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and which is able to initiate transcription of a downstream gene.
  • a promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition.
  • a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule.
  • a “recombinant nucleic acid” or “recombinant nucleotide” refers to a molecule that is constructed by joining nucleic acid molecules, which optionally may self-replicate in a live cell.
  • Retron refers to a specific type of naturally-occuring and distinct DNA sequence found in the genome of many bacteria which typically encodes three distinct components, namely, (a) a non-coding RNA (“ncRNA”) (comprising continguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function. Retrons are particularly defined by their unique ability to produce a satellite DNA known as msDNA (multicopy single-stranded DNA).
  • msDNA multicopy single-stranded DNA
  • ncRNA comprising the msr and msd elements
  • ret gene are transcribed as a single polycitronic RNA transcript which processed into the ncRNA transcript and a transcript encoding the ret gene.
  • the ncRNA then becomes folded into a specific secondary structure.
  • the RT then binds the folded ncRNA and reverse transcribes the msd region to form a single strand of cDNA (the msDNA) that remains covalently attached to the RNA template via a 2’-5 ⁇ phophodiester bond and base-pairing between the 3 ⁇ ends of the msDNA and the RNA template.
  • Retron component refers to a distinct element or feature of a retron, namely (a) a non-coding RNA (“ncRNA”) (comprising continguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function.
  • ncRNA non-coding RNA
  • RT reverse transcriptase-coding gene
  • RNA-guided nuclease is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.”
  • RNA-guided nuclease or “RNA-guided endonuclease” refers to a nuclease that associates covalently or non- covalently with a guide RNA thereby forming a complex between the guide RNA and the RNA- guided nuclease.
  • the guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence.
  • sequence identity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11- 17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna. CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference.
  • Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).
  • polypeptide including anywhere in this specification, including in Table A and the Examples
  • a reference amino acid sequence such as a polypeptide at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to another amino acid sequence (a reference amino acid sequence), it is advantageous that in the polypeptide having a percent identity to the reference amino acid sequence conserved regions of the reference amino acid sequence (e.g., conserved when compared with other retron
  • ⁇ - helical recognition lobe REC
  • NUC nuclease lobe
  • WED Wedge
  • REC ⁇ -helical recognition lobe
  • PI PAM-interacting
  • RuvC nuclease Bridge Helix
  • BH Bridge Helix
  • c one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains and/or that the polypeptide recognizes or binds a guide RNA or ncRNA as the case may be.
  • nucleic acid sequence or molecule having a percent identity with respect to a nucleic acid sequence having a percent identity with respect to another nucleic acid sequence or molecule such as a nucleic acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to another nucleic acid sequence, it is advantageous that in the nucleic acid sequence that has a percent identity to the reference nucleic acid sequence that conserved regions of the reference nucleic acid sequence (e
  • the term“subject” refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non- human subject. The subject may be of either sex and at any stage of development.
  • stem refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, formed by inverted repeat sequences connected at a “tip,” where the more 5 ⁇ or “upstream” strand of the stem bends to allows the more 3 ⁇ or “downstream” strand to base-pair with the upstream strand.
  • the number of base pairs in a stem is the “length” of the stem.
  • the tip of the stem is typically at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides.
  • An otherwise continuous stem may be interrupted by one or more bulges as defined herein.
  • the number of unpaired nucleotides in the bulge(s) are not included in the length of the stem.
  • the position of a bulge closest to the tip can be described by the number of base pairs between the bulge and the tip (e.g., the bulge is 4 bps from the tip).
  • the position of the other bulges (if any) further away from the tip can be described by the number of base pairs in the stem between the bulge in question and the tip, excluding any unpaired bases of other bulges in between.
  • Synthetic or artificial nucleic acid refers nucleic acids that are non-naturally occurring sequences. Such sequences do not originate from, or are not known to be present in any living organism (e.g., based on sequence search in existing sequence databases). Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
  • Engineered nucleic acid constructs of the present disclosure such as the engineer ed retron described herein, may be encoded by a single molecule (e.g., encoded by or present on the same plasmid or other suitable vector) or by multiple different molecules (e.g., multiple independently-replicating vectors).
  • a “target site” as used herein is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site or specific locus (“target site” or “target sequence”) targeted by a recombinant retron genome modification system disclosed herein.
  • target site a site or specific locus
  • a target sequence is the sequence to which the guide sequence of a guide nucleic acid (e.g., guide RNA) will hybridize.
  • the target site (or target sequence) 5 ⁇ -GTCAATGGACC-3 ⁇ (SEQ ID NO:19933) within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5 ⁇ -GGTCCATTGAC-3 ⁇ (SEQ ID NO:19934).
  • Suitable hybridization conditions include physiological conditions normally present in a cell.
  • the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand.”
  • Treatment As used herein, the terms“treatment,” “treat,” and“treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
  • treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
  • upstream and downstream are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5 ⁇ -to-3 ⁇ direction.
  • a first element is said to be upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5 ⁇ to the second element.
  • a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3 ⁇ to the second element.
  • variant retron RT is retron RT comprising one or more changes in amino acid residues as compared to a wild type retron RT amino acid sequence.
  • variant retron RT encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence.
  • vector permits or facilitates the transfer of a polynucleotide from one environment to another. It is a replicon such as a plasmid, phage, or cosmid into which another DNA segment may be inserted so as to bring about the replication of the inserted segment (e.g., the subject engineered retron). Generally, a vector is capable of replication when associated with the proper control elements.
  • vector may include cloning and expression vectors, as well as viral vectors and integrating vectors.
  • Wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • DETAILED DESCRIPTION [00262] The present disclosure provides systems, methods and compositions used for precise genome editing, including installing nucleic acid insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on novel and/or modified retrons or components thereof, such as modified versions of the retron RTs of Table X, modified versions of the ncRNAs of Table A, and modified versions of the RTs of Table B.
  • the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron.
  • Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non-contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase.
  • the retron that becomes modifed with the one or more genetic modifications is a naturally occurring retron or retron component (e.g., naturally occuring ncRNA of Table A or RT) ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template.
  • a naturally occurring retron or retron component e.g., naturally occuring ncRNA of Table A or RT
  • HDR homology-dependent recombination
  • the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron-related sequence provided by Table X (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see Examples).
  • the recombinant retrons are based on introducing the one or more genetic modifications into previously available retron sequences (e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.
  • retron sequences e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA
  • the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT).
  • the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems).
  • recombinant retron components e.g., recombinant ncRNA and/or recombinant RT
  • programmable nucleases e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs,
  • the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems.
  • the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions.
  • the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles.
  • the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., ncRNA and mRNA delivered by LNPs
  • protein e.g., virus-like particles
  • RNP ribonucleoprotein
  • each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • an all-RNA system e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit.
  • a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit.
  • the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems.
  • the disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems.
  • Described herein are engineered retrons comprising one or more heterologous nucleic acids.
  • the one or more heterologous nucleic acids may be inserted, for example, at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus.
  • the engineered retrons have structural improvements over their naturally existing counterparts or wild-type retrons at least with respect to the encoded ncRNA and/or the reverse transcriptase (RT), such that the engineered retron or the encoded ncRNA thereof, when delivered to a host cell, such as a mammalian host cell, exhibits various functional improvements over its naturally existing/wild- type retron elements.
  • RT reverse transcriptase
  • Exemplary (non-limiting functional improvements) may include any one or more of the features described herein.
  • the engineered retron may comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of the ncRNA encoded by the engineered retron (e.g., encoded by the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits or antagonizes) a function of the msDNA; and/or iv) modulates (e.g., improves
  • a sequence modification
  • the engineered retron is an engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), said first polynucleotide comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; and [00268] b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus.
  • ncRNA non-coding RNA
  • the engineered nucleic acid construct may further comprise a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.
  • RT reverse transcriptase
  • the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in any one of Table C.
  • RT reverse transcriptase
  • the RT does not comprise a polypeptide listed in Table X.
  • the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; and/or ii) a reverse transcriptase (RT) or a functional
  • the polynucleotide encoding the RT does not comprise a polynucleotide of Table X.
  • the engineered retron of the invention encodes an ncRNA comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA in Table B.
  • the engineered retron of the invention encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above.
  • the ncRNA may comprise: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B.
  • the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, the RT does not comprise a polypeptide listed in Table X; OR (B) i) a polynucleotide listed in Table
  • the engineered nucleic acid construct comprises: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein said msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or to resemble a secondary structure of a wild- type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) any one of the sequences and/or structures as depicted in any
  • the engineered nucleic acid construct may comprise one or more sequence modifications (e.g., an insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: a) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); b) modulates (e.g., reduces) immunogenicity of ncRNA encoded by the engineered retron (e.g., the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); c) modulates (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or d) modulates (e.g., improves) efficiency of
  • the engineered nucleic acid construct (e.g., the engineered retron) is engineered based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) the sequence of any one of Table B ncRNA sequences and/or the structure depicted in any one of FIGs.2-27; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally
  • Another aspect of the disclosure provides a vector system comprising a vector comprising the engineered retron described herein.
  • Another aspect of the disclosure provides an isolated host cell comprising the engineered retron described herein, or the vector system described herein.
  • Another aspect of the disclosure provides a pharmaceutical composition comprising the engineered retron described herein, or the vector system described herein.
  • Another aspect of the disclosure provides a delivery vehicle comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, the vector or vector system described herein, the host cell described herein, or the pharmaceutical composition described herein.
  • kits comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, and optionally instructions for genetically modifying a cell using the engineered retron described herein or the ncRNA encoded by the engineered retron described herein.
  • Another aspect of the disclosure provides a method of modifying a target DNA sequence in a host cell (e.g., a mammalian cell), the method comprising introducing into the host cell (e.g., the mammalian cell) the engineered retron of the invention, the ncRNA encoded by the engineered retron of the invention, or the vector / vector system described herein, to allow the production of the msDNA in the host cell (e.g., mammalian cell), wherein at least a part of the heterologous nucleic acid in the msDNA is integrated into the genome of the host (e.g., mammalian) cell at the target DNA sequence.
  • a host cell e.g., a mammalian cell
  • the target sequence is recognized by a suitable nuclease, such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE), and a double-stranded break (DSB) is created by the nuclease to facilitate / promote the insertion of the part of the heterologous nucleic acid into the target sequence.
  • a suitable nuclease such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)
  • DSB double-stranded break
  • the target sequence modified / inserted by the part of the heterologous nucleic acid can no longer be recognized by the nuclease to re-create a DSB.
  • Another aspect of the disclosure provides a use of the engineered retron in the various methods described herein.
  • a genome editing system comprising: a) nuclease capable of acting at a target site on a genome (e.g. human genome), such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE); and b) an engineered retron described herein, or an ncRNA encoded thereby, or a vector or a vector system comprising or encoding the same.
  • a genome editing system comprising: a) nuclease capable of acting at a target site on a genome (e.g. human genome), such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonu
  • the nuclease may be linked to one or more element(s) of the engineered retron or the encoded ncRNA.
  • the nuclease may be linked (e.g., fused or conjugated) to the reverse transcriptase of the engineered retron described herein.
  • the nuclease may engage/bind to form a complex with a nucleic acid guide sequence (such as a single-guided RNA of a Cas enzyme), wherein the guide sequence is linked to the ncRNA and/or msDNA of the engineered retron described herein.
  • Another aspect of the disclosure provides an enhanced genome editing system, comprising the genome editing system of the disclosure connected to a biomolecule that modulates host DNA repair, in order to, for example, modulate (e.g., enhance) the incorporation of the heterologous nucleic acid sequence into a genome (e.g., human genome).
  • a biomolecule that modulates host DNA repair in order to, for example, modulate (e.g., enhance) the incorporation of the heterologous nucleic acid sequence into a genome (e.g., human genome).
  • Retrons are originally discovered in 1984 in Myxococcus xanthus bacterium when a short, multi-copy single-stranded DNA (msDNA) that is abundantly present in the bacterial cell was identified. Since then, a number of naturally existing retrons have been found in many prokaryotes such as bacteria.
  • msDNA multi-copy single-stranded DNA
  • retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT).
  • ncRNA non-coding RNA
  • RT specialized reverse transcriptase
  • the retron ncRNA is the precursor of the hybrid molecule that eventually forms, and it initially folds into a typical RNA secondary structure that is recognized by the accompanying RT.
  • the translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region.
  • the RT initiates reverse transcription of the RNA towards its 5 ⁇ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA.
  • G conserved guanosine
  • a portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus.
  • cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA.
  • the msDNA remains covalently attached to the RNA template via the 2’-5 ⁇ phosphodiester bond, and base-pairs with the RNA template using the 3 ⁇ end of the msDNA.
  • FIG.1A for a general or typical organization of the retron coding sequence, including the RT encoding sequence and the msr and msd loci, as well as the synthesis of the msDNA by reverse transcription of the initial ncRNA transcript.
  • Many retrons also contain an accessory protein (not depicted in FIG.1A), which may have a variable function that may not be fully understood.
  • the engineered retrons described herein do not comprise the accessory protein naturally associated with the wild-type or template retron.
  • Applicant has discovered, analyzed, and phylogenetically classified 7257 previously unknown retrons from nature based on multiple criteria, including sequence homology and conserved predicted secondary structures, and has grouped these retrons into different phylogenetic clades based on sequence homology and/or conserved predicted secondary structures.
  • Type IA_IIA1 (FIG.2), Type 1B1 (FIG.3), Type IB2 (FIG.4), Type 1C (FIG.5), Type IIA1 other (FIG.6), Type IIA2 (FIG.7), Type IIA3 (FIG.8), Type IIA4 (FIG.9), Type IIA5 (FIG.10), Type IIIA1 (FIG.11), Type IIIA2 (FIG.12), Type IIIA3 (FIG. 13), Type IIIA4 (FIG.14), Type IIIA5 (FIG.15), Type IIIunk (FIG.16), Type IV (FIG.
  • FIG.1B.1 depicts an embodiment of a recombinant retron construct (e.g., a nucleotide sequence cloned into an expression vector) contemplated by the present disclosure.
  • the single thin black line represents a double-stranded nucleotide sequence (e.g., as cloned into an expression vector, such as a plasmid).
  • the recombinant retron is constructed by modifying a starting point retron DNA sequence encoding a ncRNA (the msr/msd region) (such as any one of the herein disclosed 7257 newly discovered retron sequences, and specifically any one of the 7257 ncRNA sequences of Table B.
  • a starting point retron DNA sequence encoding an ncRNA may be modified in any number of ways and can including one modification or more than one modification.
  • the retron DNA may modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
  • nucleotide of a starting point retron e.g., a wildtype retron
  • the nucleotides may be contiguous or non-contiguous.
  • an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature.
  • an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic / artificial / recombinant nucleic acid sequences.
  • an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences.
  • modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci.
  • Linking guide RNA molecules to the 5 ⁇ and/or 3 ⁇ ends also represents another modification contemplated by the recombinant retrons disclosed herein.
  • the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons. These modifications are depicted in FIG.1B.
  • the DNA encoding the RT may also be modified to obtain a recombinant RT.
  • the RT-encoding DNA may modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95
  • Such modifications to the DNA encoding ncRNA and/or RT may modulate the function of the ncRNA and/or RT in various ways, including i) modulating (e.g., enhancing) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulating (e.g., reducing) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) modulating (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or iv) modulating (e.g., improving) efficiency of targeted genome editing / engineering.
  • modulating e.g., enhancing
  • reverse transcription e.g., processivity, accuracy/fidelity, and/or production of the msDNA
  • the present disclosure provides recombinant retrons having the general structure of: a) an msr locus; b) an msd locus encoding the msd RNA portion of the msDNA; c) a sequence encoding a retron reverse transcriptase (RT) (optionally in trans to the ncRNA), wherein the msd RNA is capable of being reverse transcribed (e.g., in a host cell such as a mammalian cell) to form an msDNA by the retron reverse transcriptase (RT); d) a heterologous nucleic acid (e.g., heterologous DNA) capable of being transcribed with the msr locus and/or the msd locus, optionally, the heterologous nucleic acid is inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus.
  • RT retron reverse transcriptas
  • the engineered retrons of the invention are optionally structurally further modified to include one or more heterologous nucleic acids.
  • the engineered retron may be further modified to provide various functional improvements, such as (without limitation), to enhance the production of msDNA in a cell (e.g., a mammalian cell, including a human cell).
  • a cell e.g., a mammalian cell, including a human cell.
  • the disclosure provides engineered retrons based on their conserved predicted secondary structures, such as those in FIGs.2-27.
  • the disclosure provides engineered retrons based on their sequence identity. Exemplary RT amino acid sequences and ret gene nucleic acid sequences are provided in Table A.
  • RT consensus amino acid sequences and/or ret gene nucleic acid sequences are provided in Table C.
  • Exemplary ncRNA sequences are provided in Table B.
  • Retron sequences that may be provisoed out of the scope of the invention in some embodiments are provided in Table X.
  • exemplary engineered retrons of the invention (1) are engineered based on or engineered to resemble the secondary structures as depicted in any one of FIGs.2-27, and/or (2) are provided in Table B.
  • Sequences with significant sequence percentage identity are also within the scope of the invention.
  • sequence percentage identity e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity) are also within the scope of the invention.
  • the engineered nucleic acid construct comprises: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein said msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or to resemble a secondary structure of a wild- type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) any one of the sequences and/or structures as depicted in any one
  • insertion of a heterologous nucleic acid includes deletion of a retron nucleic acid.
  • the inserted heterologous nucleic acid substitutes for a retron nucleic acid which is deleted. There is no requirement that the inserted heterologous nucleic acid and the deleted retron nucleic acid be of the same or similar size.
  • substitution comprises replacment of a portion of a hairpin loop region of a retron.
  • substitution comprises replacment of a portion of a stem-loop region of a retron.
  • substitution comprises replacement of a region of a retron that does not comprise a stem or a loop.
  • identification of retron structure involves comparison to model ncRNA structures provided herein, e.g. by comparison to one or more of Figs.2-27.
  • identification of retron structure involves modeling by an nucleic acid folding algorithm, such as but not limited to RNAfold (Gruber AR, et al., The Vienna RNA Websuite. Nucleic Acids Research, Volume 36, Issue suppl_2, 1 July 2008, Pages W70-W74), UNAFold (Markham NR, et al., UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol.
  • the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%
  • the RT does not comprise a polypeptide identified in Table X.
  • the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide of Table A and/or ii) a consensus poly
  • the polynucleotide encoding the RT does not comprise a polynucleotide identified in Table X.
  • the engineered retron of the invention encodes an ncRNA comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA of Table B.
  • Engineered ncRNAs of the invention can diverge in size from ncRNAs on which they are based and the proportion of the the ncRNA retained in the engineered ncRNA can vary.
  • the retained amount of the ncRNA is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or from 50% to 80%, or from 60% to 85%, of from 70% to 90%, or from 80% to 95%, or from 85% to 98%, or from 90% to 99%, or all of the ncRNA.
  • the engineered retron of the invention encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above.
  • the ncRNA may comprise: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B;
  • the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, the RT does not comprise a polypeptide identified in Table X; OR (B) i) a polynucleotide listed in
  • the heterologous nucleic acid is between >20 nucleotides and about 10,000 nucleotides.
  • the engineered retron may further comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits, either permanently or transiently) a function of the engineered retron in a host (e.g.
  • Retron msr gene, msd gene, and RT nucleic acid sequences e.g., the ret gene
  • the encoded retron reverse transcriptase protein sequences that may serve as the template of the engineered retron described herein may be derived from any source, such as those in Table A, optionally excluding those associated with the sequences of Table X.
  • template or wild-type (wt) sequences of the msr gene, msd gene, and the RT coding sequence (viz., the ret gene) used in the engineered retron are derived from a bacterial retron.
  • representative template/wild-type retrons are from gram negative bacteria.
  • the retron is from a bacterium listed in Table X.
  • the engineered retrons are engineered based on clades defined on retron/retron RTs, in which the retrons are associated with a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions.
  • clades are based primarily upon naturally occurring ncRNA and retron/retron RT, and an additional protein or RT-fused domain, the clades, for the purpose of serving as the templates for the subject engineered retrons, are not limited to naturally occurring sequences.
  • retrons may be considered phylogenetically related based on a Neighbor-Joining algorithm of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the retron RT.
  • retrons may be considered phylogenetically related when / if the same RT, or closely related RT, can recognize the secondary structures of the ncRNA of the retrons and reserve transcribe the retrons to produce msDNA.
  • sequence alignments between different retron sequences e.g., ncRNA and/or RT (protein and/or nucleic acid) sequences
  • secondary structure generations are based on software known to one of ordinary skill in the art.
  • the retron ncRNA sequences including msr and msd sequences within the same clade may be highly conserved at certain positions, while being less conserved at other positions.
  • Exemplary consensus sequences based on clade members were generated (see, for example, the corresponding FIGs.2-27, respectively) to show these conserved sequences and/or secondary structures, including the highly conserved nucleotides with at least 97% sequence conservation at red lettered-nucleotides, those with between 90-97% sequence conservation at black lettered-nucleotides, and 75-90% nucleotide sequence identity at grey-lettered nucleotides.
  • the template ncRNA based on which the subject engineered retron is modified is a consensus sequence for the various retron ncRNA (including msr and msd nucleic acid sequences) clades, as provided in any one of SEQ ID NOs: of Table B and the corresponding FIGs.2-27, respectively, including the bases that are highly conserved and depicted by a specific-colored letters or circles, and optionally further including bases that may be present at specific locations by specific-colored circles.
  • the engineered retron is engineered, based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: 1) the sequences and/or structures as depicted in any one of SEQ ID NOs: of Table B and the corresponding FIGs.2-27, respectively; or 2) a variant of 1), having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered- nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered- nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered- nucleotides.
  • A) up to 1, 2, or 3 e.g., up to 1) nucleotide changes per 10 red lettered- nucleotides
  • the variant of 1) further comprises: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; b) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; c) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or d) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides.
  • the engineered retron may be engineered by introducing the sequence modifications (e.g., deletions, additions, or substitutions) into the wild-type retron encoding wild-type retron ncRNA, or into the retron encoding the consensus retron ncRNA.
  • a variant retron may not satisfy the sequence and/or structural requirements of any one of SEQ ID NOs: of Table B and the corresponding FIGs.2-27, respectively, but may still be a suitable template for the engineered retron described herein, so long as one or more of the conditions set forth in A) – C) and/or a)-d) are met.
  • the highly conserved sequences in the template retrons are preserved / conserved or substantially preserved / conserved in the engineered retron described herein.
  • all or substantially all the red lettered-nucleotides i.e., those conserved in about 97% or more of the retrons in the same clade
  • no more than 1, 2, or 3 e.g., up to 1 nucleotide change(s) (e.g., deleted or substituted) occur per 10 red lettered-nucleotides in the engineered retron described herein.
  • red lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.
  • all or substantially all the black lettered-nucleotides i.e., those conserved in about 90-97% of the retrons in the same clade) are preserved / conserved in the engineered retron described herein.
  • nucleotide change(s) e.g., deleted or substituted
  • nucleotide change(s) e.g., deleted or substituted
  • no more than about 3%, 4%, 5% or 10% of the black lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.
  • all or substantially all the grey lettered-nucleotides are preserved / conserved in the engineered retron described herein.
  • no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 e.g., up to 3 or 4, or up to 7, 8, or 9 nucleotide change(s) (e.g., deleted or substituted) occur per 10 grey lettered-nucleotides are changed in the engineered retron described herein.
  • no more than about 5%, 10%, 15%, 20%, or 25% of the grey lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.
  • all or substantially all the red circled-nucleotides i.e., those with a nucleotide in about 97% or more of the retrons in the same clade
  • no more than 1, 2, or 3 e.g., 0.3, 0.5, or up to 1 nucleotides are absent (e.g., deleted) per 10 red circled-nucleotides in the engineered retron described herein.
  • nucleotides are present per 10 red circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 0.3%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the red circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein. [00331] In certain embodiments, all or substantially all the black circled-nucleotides (i.e., those with a nucleotide in about 90-97% of the retrons in the same clade) are present in the engineered retron described herein.
  • nucleotides are absent (e.g., deleted) per 10 black circled-nucleotides in the engineered retron described herein.
  • 6, 7, 8, 9, or 10 e.g., 8, 9 or 10 nucleotides are present per 10 black circled-nucleotides in the engineered retron described herein.
  • no more than about 1%, 2%, 3%, 5% or 10% of the black circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.
  • all or substantially all the grey circled-nucleotides are present in the engineered retron described herein.
  • no more than 1, 2, 3, 4, or 5 e.g., up to 2, 3, or 4
  • nucleotides are absent (e.g., deleted) per 10 grey circled-nucleotides in the engineered retron described herein.
  • 4, 5, 6, 7, 8, 9, or 10 e.g., 6, 7, 8, 9 or 10 nucleotides are present per 10 grey circled-nucleotides in the engineered retron described herein.
  • no more than about 5%, 10%, 15%, 20%, or 25% of the grey circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.
  • all or substantially all the white circled-nucleotides i.e., those with a nucleotide in about 50-75% of the retrons in the same clade
  • no more than 1, 2, 3, 4, 5, 6, or 6 are absent (e.g., deleted) per 10 white circled-nucleotides in the engineered retron described herein.
  • 2, 3, 4, 5, 6, 7, 8, 9, or 10 e.g., 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present per 10 grey circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the white circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein. [00334] In some embodiments, the engineered retron is synthetically produced.
  • the synthetically produced engineered retron comprises the sequences and/or secondary structures as depicted in any one of SEQ ID NOs: of Table B and the corresponding FIGs.2-27, respectively, and at least the conserved color lettered nucleotides according to their respective levels of sequence identity (e.g., red, black and gray letters), and/or at least the conserved colored circle nucleotides according to their respective levels of probability of sequence presence (e.g., red, black and gray circles).
  • the sequence modification in the engineered retrons leads to/results in the encoded retron ncRNA having the desired functional improvement.
  • the one or more sequence modifications comprises, in the ncRNA, one or more of: (i) a modified (e.g., mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii) an extension or shortening of a1, a2, or both a1 and a2; (iii) an extension or shortening of a spacer sequence between hairpin loops (e.g., S1, S2, S3, and/or S4 in FIG.2, or any of the S regions in an one of FIGs.2-27); (iv) an additional or modified (e.g., mutated or eliminated) bulge in hairpin loops (e.g., L2 and/or L3 in FIG.2, or any of the L regions in an one of FIGs.2-27 (e.g., by removing unpaired bases in the bulge, or by replacing unpaired bases with an equivalent number of base pairs)); (v) a modified (e) a modified (e.g.
  • the a1 and a2 regions are both single- stranded and substantially reverse complementary to each other, forming a stem with optional interruption by a symmetric or asymmetric bulge, with optional one or more 5 ⁇ and/or 3 ⁇ overhang/unpaired nucleotide(s), wherein the a1 region generally ends before (e.g., ends immediately 5 ⁇ to) the conserved branching guanosine (G) providing the 2’-OH for reverse transcription priming.
  • G conserved branching guanosine
  • the sequence change comprises a mutated, reduced, or eliminated bulge in the a1/a2 stem region, including sequence change(s) in one (i.e., a1 or a2) strand, or both a1 and a2 strands.
  • the sequence change comprises deleting nucleotides from a1, a2, or both a1 and a2, such that the size of the bulge is reduced, or a symmetrical bulge becomes asymmetrical or vice versa, or a bulge is eliminated.
  • the sequence change comprises replacing/substituting nucleotides in a1, a2, or both a1 and a2, such that previously unpaired bases in the bulge become base-paired.
  • the sequence change comprises replacing an unpaired purine base with one or more unpaired pyrimidine base(s).
  • the sequence change comprises replacing an unpaired pyrimidine base with one or more unpaired purine base(s).
  • the sequence change comprises replacing one unpaired purine base (e.g., A or G) with another unpaired purine base (e.g., G or A, respectively).
  • the sequence change comprises replacing one unpaired pyrimidine base (e.g., T/U or C) with another unpaired pyrimidine base (e.g., C or T/U, respectively).
  • the sequence change comprises an extension or shortening of a1, a2, or both a1 and a2.
  • the length of a1 can be shortened by deleting 5 ⁇ overhang, deleting any upstream bulge nucleotides, deleting bases involved in base-pairing.
  • the length of a1 can be extended by adding 5 ⁇ overhang, adding any upstream bulge nucleotides, adding bases involved in base-pairing.
  • the length of a2 can be shortened by deleting 5 ⁇ overhang, deleting any downstream bulge nucleotides, deleting bases involved in base-pairing.
  • the length of a2 can be extended by adding 5 ⁇ overhang, adding any downstream bulge nucleotides, adding bases involved in base-pairing.
  • the spacer sequences between hairpin loops as depicted in any one of FIGs.2-27, can be extended or shortened.
  • the modification can be by inserting a heterologous nucleic acid sequence in spacer sequences between hairpin loops (e.g., S1, S2, S3 and/or S4 in FIG.2).
  • a heterologous nucleic acid sequence is inserted in a spacer sequence in the msd region.
  • the modification of the spacer region can be by interrupting the spacer with additional bulges or hairpin loops.
  • a bulge in the hairpin loops are mutated or eliminated (e.g., by removing unpaired bases) in the bulge, such that, for example, a symmetric bulge becomes an unsymmetrical bulge, or an unsymmetrical bulge becomes a symmetric one or an even more unsymmetrical one.
  • unpaired bases in the bulge is replaced with an equivalent number of base pairs. The additional base pairs may be merged into the stem at one or both ends of the previous bulge, or may bisect a previous bulge to create two bulges.
  • the length of one or more hairpin loops as depicted in any one of FIGs.2-27, can be extended or shortened.
  • the number of unpaired bases within the tip or loop can be increased or decreased.
  • a heterologous nucleic acid sequence of interest can be inserted within the tip or the hairpin loop.
  • the heterologous nucleic acid sequence of interest is inserted within the tip or the hairpin loop in the msd locus.
  • the GC content in the tip or hairpin loops are increased or decreased.
  • a hairpin loop can be deleted.
  • the ncRNA with the 5 ⁇ end and the 3 ⁇ end of the ncRNA can be circularized by being connected either directly, or via a spacer sequence.
  • one or more hairpin loops e.g. L1, L2, L3 and/or L4 of FIG.2 are modified to have complement, reverse, or reverse complement sequences.
  • the branching guanosine (G) capable of initiating reverse transcription priming is repositioned.
  • the G can be placed further downstream of the end of the a1 sequence by, for example, 1, 2, 3, 4, or 5 additional nucleotides.
  • the one or more heterologous nucleic acid sequences comprise: a) a heterologous nucleic acid (such as the coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as in an S region (e.g., S1, S2, S3 and/or S4 in FIG.2, or any of the S regions in any one of FIGs.2-27), or the tip of an L region (e.g., L1, L2, L3 and/or L4 in FIG.2, or any of the L regions in any one of FIGs.2-27), or upstream or downstream of either the msr locus or the
  • an antisense sequence complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid can be included, wherein the antisense sequence hybridizes to and inhibits the gRNA in the encoded retron ncRNA, and wherein the antisense sequence is removed upon reverse transcription of the msDNA.
  • gRNA CRISPR/Cas guide RNA
  • said heterologous nucleic acid encodes a protein or peptide of interest, or wherein said heterologous nucleic acid comprises or encodes a donor / template sequence (e.g., a donor that corrects / repairs / removes a mutation at the target genome site, such as a mutated exon in a disease gene; a functional DNA element (such as a promoter, an enhancer, a protein binding sequence, a methylation site, a homology region for assisting gene editing, etc.); or a coding sequence for a functional RNA element (ncRNAs, etc.)).
  • a donor / template sequence e.g., a donor that corrects / repairs / removes a mutation at the target genome site, such as a mutated exon in a disease gene
  • a functional DNA element such as a promoter, an enhancer, a protein binding sequence, a methylation site, a homology region for assisting gene editing, etc.
  • the protein or peptide of interest comprises a therapeutic protein (such as a wildtype protein defective in a disease cell, or a therapeutic antibody or antigen-binding fragment thereof) useful in treating a disease.
  • a therapeutic protein such as a wildtype protein defective in a disease cell, or a therapeutic antibody or antigen-binding fragment thereof.
  • Other heterologous nucleic acids of the invention are described in other section of the specification, all incorporated herein by reference.
  • the template/wild-type retron for the engineered retron encodes a wild-type or consensus retron ncRNA polynucleotide having a consensus secondary structure shown in any one of FIGs.2-27, which are described individually below:
  • Variants of this template include a variant having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in SEQ ID NO. XX and FIGs.2-27.
  • the non-coding RNA (ncRNA) portion of the engineered retron comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least
  • the ncRNA does not comprise an ncRNA associated with the sequences of Table X.
  • Amplification of an engineered retron described herein may be performed, for example, before transfection of cells or ligation into vectors. Any method for amplifying the engineered retron may be used, including, but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription mediated amplification
  • SDA strand displacement amplification
  • LCR ligase chain reaction
  • the engineered retron comprise common 5 ⁇ and 3 ⁇ priming sites to allow amplification of retron sequences in parallel with a set of universal primers.
  • a set of selective primers is used to selectively amplify a subset of retron sequences from a pooled mixture.
  • the template/wild-type retron for the engineered retron encodes a wild-type or consensus retron ncRNA polynucleotide having a consensus secondary structure shown in FIG.2-27, and as described individually below: Type IA/IIA1 retron (FIG.2)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-a2-S4-L4, wherein: a1/a2 is a stem 8 bp in length; L1 is a stem of 5 bps with a 10-nt tip; L2 is a stem of 7 bps with a 5-nt tip,
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.2.
  • Type IB1 retron (FIG.3)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 6 bps in length with a 2/2 bulge 3 bps from the tip, wherein a1 has a 2-nt overhang and a2 has a 6-nt overhang; L1 is a stem of 14 bps with a 3-nt tip, a 1/0 bulge 4 bps from the tip, and a 0/6 bulge 10 bps from the tip; L2 is a stem of 23 bps with a 5-nt tip, a 1/1
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.3.
  • Type IB2 retron (FIG.4)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-L4-S4-a2, wherein: the a1/a2 stem is 16 bp in length, with a 17-base 5 ⁇ overhang and a 16-base 3 ⁇ overhang; L1 is a stem of 6 bps with a 4-nt tip; L2 is a stem of 4 bps with a 4-nt tip, with a 2/2 bulge 2 nts from the tip; L3 is a stem of 3 bps with a 5-nt tip; L4 is a stem of
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.4.
  • Type 1C retron (FIG.5)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 13 bps in length; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 10 bps with a 5-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleot
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.5.
  • Type IIA1 retron (FIG.6)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 10 bps in length with a 1-nt overhang on a2; L1 is a stem of 10 bps with a 3-nt tip; L2 is a stem of 7 bps with a 5-nt tip; L3 is a stem of 27 bps with a 8-nt tip and a 0/2 bulge 26 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.6.
  • Type IIA2 retron (FIG.7)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-L3-S3-a2, wherein: a1/a2 is a stem 7 bp in length with no overhangs; L1 is a stem of 8 bps with a 3-nt tip; L2 is a stem of 30 bps with a 8-nt tip, a 1/1 bulge 2 bps from the tip, and a 1/1 bulge 27 bps from the tip; L3 is a stem of 8 bps with a 5-nt tip, and a 0/1 bulge 3 nt from the tip
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.7.
  • Type IIA3 retron (FIG.8)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a stem 6 bps in length; L1 is a stem of 8 bps with a 9-nt tip; L2 is a stem of 8 bps with a 3-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L2 and L3; S3 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides as denoted by the colored letters
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.8.
  • Type IIA4 retron (FIG.9)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-a2-L1-S1-L2-L3-S2-L4-S3, wherein: a1/a2 is a stem 3 bp in length with no overhangs and a 7-nt tip; L1 is a stem of 7 bps with a 3-nt tip; L2 is a stem of 6 bps with a 4-nt tip; L3 is a stem of 40 bps with a 5-nt tip, and a 2/2 bulge 3 bps from the tip, a 5/4 bulge 10 bps from the tip, and a 12/15 bulg
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.9.
  • Type IIA5 novel retron FIG.10
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 15 bps in length with a 1-nt overhang on a1, a 13-nt overhang on a2, and a 7/5 bulge 13-nt from the tip; L1 is a stem of 10 bps with a 3-nt tip; L2 is a stem of 35 bps with a 3-nt tip; L3 is a stem of 6 bps with a 5-nt tip; S1 is a single-strand
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.10.
  • Type IIIA1 retron (FIG.11)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a stem 2 bps in length with a 1-nt overhang on a2; L1 is a stem of 8 bps with a 4-nt tip; L2 is a stem of 9 bps with a 3-nt tip and a 1/1 bulge 3 bps from the tip; L3 is a stem of 20 bps with a 3-nt tip and a 1/2 bulge 3 bps from the tip; S1 is a single-stranded spacer
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.11.
  • Type IIIA2 retron (FIG.12)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-L4-S5-a2, wherein: a1/a2 is a stem 15 bp in length; L1 is a stem of 6 bps with a 4-nt tip; L2 is a stem of 13 bps with a 5-nt tip; L3 is a stem of 4 bps with a 8-nt tip; L4 is a stem of 20 bps with a 4-nt tip and a 2/2 bulge 6 bp from the tip; S1 is a single
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.12.
  • Type IIIA3 retron (FIG.13)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-L4-L5-L6-S5-a2, wherein: a1/a2 is a stem 24 bps in length and having a 1/0 bulge 15 bps from the tip, and a 1/1 bulge 19 bps from the tip; L1 is a stem of 7 bps with a 4-nt tip; L2 is a stem of 9 bps with a 8-nt tip; L3 is a stem of 8 bps with a 4-nt tip; L4 is a wild-type retron ncRNA polynucleo
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.13.
  • Type IIIA4 retron (FIG.14)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 5 bps in length with a 1/2 bulge 2 bps from the tip; L1 is a stem of 8 bps with a 6-nt tip; L2 is a stem of 8 bps with a 5-nt tip; L3 is a stem of 13 bps with a 14-nt tip and a 1/0 bulge 2 bps from the tip; S1 is a single-stranded spacer region between a1/a
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.14.
  • Type IIIA5 retron (FIG.15)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-L3-L4-S3-a2, wherein: the a1/a2 stem is 11 bp in length with no overhang; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 14 bps with a 5-nt tip; L3 is a stem of 9 bps with a 7-nt tip; L4 is a stem of 15 bps with a 7-nt tip; S1, S2, and S3 are single-stranded spacer regions between the a1/
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.15.
  • Type IIIunk retron (FIG.16)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 11 bps in length; L1 is a stem of 12 bps with a 2-nt tip; L2 is a stem of 21 bps with a 1-nt tip; L3 is a stem of 20 bps with a 4-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.16.
  • Type IV retron (FIG.17)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a stem 9 bp in length with no overhang; L1 is a stem of 5 bps with a 6-nt tip; L2 is a stem of 9 bps with a 4-nt tip; L3 is a stem of 26 bps with a 5-nt tip, a 0/1 bulge 7 bps from the tip, and a 0/1 bulge 9 bps from the tip; S1 is a single-stranded spacer region between the
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.17.
  • Type IX retron (FIG.18)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-L1-S1-L2-S2-a2, wherein: a1/a2 is a stem 12 bp in length, wherein a1 has a 14-nt overhang and a2 has a 2-nt overhang; L1 is a stem of 11 bps with a 3-nt tip and a 1/3 bulge 7 bp from the tip; L2 is a stem of 25 bps with a 7-nt tip; S1 is a single-stranded spacer region between L1 and L2; S2 is a single-stranded spacer
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.18.
  • Type V retron (FIG.19)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 13 bps in length; L1 is a stem of 20 bps with a 4-nt tip and a 6/4 bulge 6 bps from the tip; L2 is a stem of 14 bps with a 4-nt tip and a 1/0 bulge 5 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.19.
  • Type VI retron (FIG.20)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-L4-S4-a2, wherein: a1/a2 is a stem 4 bp in length with a 1 bp 5 ⁇ overhang; L1 is a stem of 7 bps with a 4-nt tip; L2 is a stem of 8 bps with a 4-nt tip; L3 is a stem of 16 bps with a 6-nt tip, a 3/4 bulge 3 bps from the tip, a 2/3 bulge 5 bps from the tip, and a 3/1 bulge 8
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.20.
  • Type XI Group 1 retron (FIG.21)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 16 bps in length with a 5-nt overhang on a1, and a 3-nt overhang on a2; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 7 bps with a 13-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.21.
  • Type XI Group 2 retron (FIG.22)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 13 bps in length with a 1-nt overhang on a2; L1 is a stem of 7 bps with a 3-nt tip and a 2/2 bulge 1 bp from the tip; L2 is a stem of 8 bps with a 20-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.22.
  • Type XII retron (FIG.23)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem 13 bps in length with a 1-nt overhang on a2; L1 is a stem of 7 bps with a 3-nt tip, and a 2/2 bulge 1 bp from the tip; L2 is a stem of 8 bps with a 19-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.23.
  • Type XIII retron (FIG.24)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-L3-S3-a2, wherein: a1/a2 is a stem 7 bp in length with no overhangs; L1 is a stem of 8 bps with a 3-nt tip; L2 is a stem of 30 bps with a 8-nt tip, a 1/1 bulge 2 bps from the tip, and a 1/1 bulge 27 bps from the tip; L3 is a stem of 8 bps with a 5-nt tip, and a 0/1 bulge 3 nt from the
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.24.
  • Type XIV retron (FIG.25)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-L2-S2-L3-S3-a2, wherein: the a1/a2 stem is 15 bp in length with no overhang, and a 4/2 bulge 7 bps from the 5 ⁇ end of a1; L1 is a stem of 8 bps with a 5-nt tip; L2 is a stem of 7 bps with a 5-nt tip; L3 is a stem of 13 bps with a 2-nt tip, a 5/9 bulge 5 bps from the tip, and a 5/5 bulge 8 bps
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.25.
  • Ec107-like retron (FIG.26)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 12 bp in length; L1 is a stem of 4 bps with a 8-nt tip; L2 is a stem of 8 bps with a 3-nt tip; L3 is a stem of 22 bps with a 3-nt tip, a 4/6 bulge 6 bp from the tip, a 3/3 bulge 13 bp from the tip, and a 1/1 bulge 18 bp from the tip;
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.26.
  • Outgroup A retron (FIG.27)
  • the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem 5 bps in length with a 2-nt overhang on a2; L1 is a stem of 11 bps with a 4-nt tip; L2 is a stem of 8 bps with a 3-nt tip; L3 is a stem of 18 bps with a 3-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded space
  • the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG.27.
  • Amplification of an engineered retron described herein may be performed, for example, before transfection of cells or ligation into vectors. Any method for amplifying the engineered retron may be used, including, but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription mediated amplification
  • SDA strand displacement amplification
  • LCR ligase chain reaction
  • the engineered retron comprise common 5 ⁇ and 3 ⁇ priming sites to allow amplification of retron sequences in parallel with a set of universal primers.
  • a set of selective primers is used to selectively amplify a subset of retron sequences from a pooled mixture.
  • Variants of these templates include a variant having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; b) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; c) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9
  • the non-coding RNA (ncRNA) portion of the engineered retron comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B.
  • a polynucleotide e.g., a DNA molecule
  • the ncRNA does not comprise an ncRNA associated with the RT sequences of Table X. Format of ncRNA and guide RNA [00423]
  • the ncRNA and the guide RNA can be delivered as a single molecule, i.e., with the guide RNA fused to the 5’ and/or 3’ end of the ncRNA.
  • the ncRNA may have guide RNAs located at both ends in some embodiments.
  • the guide RNA and ncRNA may be provided and/or delivered as separate components. As shown in Example 4, separation of the guide RNA from the ncRNA can result in increased editing efficiency.
  • ncRNA-gRNA fusion may be co-delivered with a separate guide RNA.
  • Modified ncRNAs [00426] In still other embodiments, the ncRNAs disclosed herein may be modified by introducing additional RNA motifs into the ncRNAs, e.g., at the 5 ⁇ and 3 ⁇ termini of the ncRNAs, or even at positions therein between (e.g., in the msr or msd regions) to improve transcriptional production and/or stability and/or function (e.g., RT-DNA production).
  • RNA hairpins may include, but are not limited to RNA hairpins, RNA step-loops, RNA quadruplexes, cap structures, and poly(A) tails, or ribozyme functions and the like.
  • ncRNAs could also be modified to include one or more nuclear localization sequences.
  • Additional RNA motifs could also improve RT processivity of the ncRNA or enhance ncRNA activity by enhancing RT binding. Addition of dimerization motifs - such as kissing loops or a GNRA tetraloop/tetraloop receptor pair - at the 5 ⁇ and 3 ⁇ termini of the ncRNA could also result in effective circularization of the ncRNA, improving stability.
  • ncRNAs could be further improved via directed evolution, in an analogous fashion to how protein function can be improved.
  • RNAs including the guide RNAs and the ncRNAs used in the compositions of the disclosure have undergone a chemical or biological modification to render them more stable.
  • exemplary modifications to an RNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base.
  • RNA modifications includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).
  • RNAs used in the compositions of the disclosure include, but are not limited to, 4'- thio-modified bases: 4'-thio-adenosine, 4'-thio-guanosine, 4'-thio-cytidine, 4'-thio-uridine, 4'- thio-5-methyl- cytidine, 4'-thio-pseudouridine, and 4'-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudour
  • modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme).
  • modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3' UTR or the 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).
  • RNAs include a 5' cap structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • Naturally occurring cap structures comprise a 7- methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside.
  • the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase.
  • the addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription.
  • the terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.
  • Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J.
  • RNA e.g., ncRNA
  • a poly A or poly U tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A or poly U tail can be added to an RNA molecule thus rendering the RNA more stable. Poly A or poly U tails can be added using a variety of art-recognized techniques.
  • long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology.1996; 14: 1252-1256).
  • a transcription vector can also encode long poly A tails.
  • poly A tails can be added by transcription directly from PCR products.
  • Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
  • the length of a poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides.
  • a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • mRNAs include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail.
  • RNAs according to the present disclosure e.g., ncRNAs
  • RNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • an appropriate RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • the ncRNAs can comprise an MS2 modification, as specific RNA hairpin structure recognized in nature by a certain MS2-binding protein. This domain can help to stabilize the ncRNA and improve the editing efficiency.
  • MS2 modification as specific RNA hairpin structure recognized in nature by a certain MS2-binding protein. This domain can help to stabilize the ncRNA and improve the editing efficiency.
  • the disclosure contemplates other similar modifications.
  • the engineered retron may comprise or encode a heterologous nucleic acid (e.g., DNA or RNA) within the msr locus or the msd locus (such as in an S region or the tip of an L region in the consensus structure in any one of FIGs.2-27, and variants thereof), or upstream or downstream of either the msr locus or the msd locus.
  • a heterologous nucleic acid e.g., DNA or RNA
  • the heterologous nucleic acid is inserted within the msd locus.
  • the heterologous nucleic acid is inserted upstream of the msr locus.
  • the heterologous nucleic acid is inserted upstream or downstream of the msd locus.
  • the heterologous nucleic acid is inserted in spacer sequences (e.g., S1, S2, S3 and/or S4 as depicted in FIG.2) between hairpin loops and/or within hairpin loops (e.g., L1, L2, L3 and/or L4 as depicted in FIG. 2), e.g., the tip or loop region, or within a bulge.
  • the heterologous nucleic acid sequence can be inserted in the tip of an L region or hairpin loop (e.g., L1, L2, L3 and/or L4 as depicted in FIG.2).
  • one or more heterologous nucleic acids are inserted into the msd locus.
  • a first heterologous nucleic acid is inserted into the msd locus, and a second heterologous nucleic acid is inserted either upstream of the msr locus or upstream or downstream of the msd locus.
  • the heterologous nucleic acid comprises flanking contiguous nucleotides (also referred to as homologous arms) that are substantially complementary to a target site genomic sequence of a cell to facilitate insertion of at least part of the heterologous nucleic acid into the genome of the cell at the target site via homology directed repair (HDR).
  • HDR homology directed repair
  • the heterologous nucleic acid is between >20 nucleotides and 10,000 nucleotides (e.g., including the flanking homologous arms).
  • one or both of the homology arm(s) on the heterologous nucleic acid are 100% identical to a target genomic sequence.
  • one or both of the homology arm(s) on the heterologous nucleic acid are less than 100% complementary to the target genomic sequence, for example, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target genomic sequence.
  • the part of the heterologous nucleic acid to be inserted into the target genome sequence is sometimes referred to as a donor sequence.
  • the donor sequence may be partially identical to the full length or portions thereof of a target genomic sequence, or may be unrelated to the target genomic sequence.
  • the donor sequence may be used, for example, to introduce modifications (e.g., substitutions, deletions, insertions, or a combination thereof) such as mutations or other genetic changes (e.g., genetic elements such as stop codons or a shift in an open reading frame on the target polynucleotide) into its target sequence.
  • the heterologous nucleic acid sequence is or encodes a biologically active molecule such as, but not limited to, a therapeutic protein.
  • a biologically active molecule such as, but not limited to, a therapeutic protein.
  • Any therapeutic proteins may be encoded by the heterologous nucleic acid.
  • the heterologous nucleic acid sequences encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors.
  • the heterologous sequences may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer.
  • TNF tumor necrosis factor
  • the heterologous sequences may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., heterologous sequences are or encode dystrophin or a functional fragment or variant thereof such as the numerous dystrophin minigenes or microdystrophine coding sequences known in the art), cardiovascular disease (e.g., heterologous sequences are or encode SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., heterologous sequences is or encodes NGF, BDNF, GDNF, NT-3, etc.).
  • muscular dystrophy e.g., heterologous sequences are or encode dystrophin or a functional fragment or variant thereof such as the numerous dystrophin minigenes or microdystrophine coding sequences known in the art
  • cardiovascular disease e.g., heterologous sequences are or encode SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd
  • the heterologous nucleic acid sequence may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer.
  • the heterologous nucleic acid sequence may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., heterologous nucleic acid sequence is or encodes Dystrophin), cardiovascular disease (e.g., heterologous nucleic acid sequence is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., heterologous nucleic acid sequence is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., heterologous nucleic acid sequence is or encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., heterologous
  • the heterologous nucleic acid sequence is or encodes a factor that can affect the differentiation of a cell.
  • the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302/367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.
  • iPS induced pluripotent stem
  • the heterologous nucleic acid sequence is or encodes a factor for transdifferentiating cells.
  • Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.
  • the heterologous nucleic acid encodes a therapeutic antibody, or an antigen-binding fragment thereof.
  • the heterologous nucleic acid encodes a protein for replacement therapy.
  • the protein may be defective in a disease cell or a disease organism / individual, and the wild-type protein or a functional fragment or variant thereof, when delivered by the heterologous nucleic acid to the disease cell / tissue / organism / individual, at least partially or fully restores the lost function of the protein the disease cell / tissue / organism / individual.
  • HNS donor DNA template
  • the heterologous nucleic acid sequence is a donor DNA template that can be integrated into a host genome via HDR.
  • the heterologous nucleic acid sequence is a donor DNA that can serve as a template or primer for recombineering during replication.
  • the heterologous nucleic acid comprises or encodes a donor / template sequence, wherein the donor / template corrects / repairs / removes a mutation at the target genome site.
  • the mutation may be a mutated exon in a disease gene.
  • the donor / template may encode or comprises a functional DNA element, such as a promoter, an enhancer, a protein binding sequence, a methylation site, or a homology region for assisting gene editing, etc.
  • donor DNA or “donor DNA template” it is meant a single-stranded DNA to be inserted at a site cleaved by a gene-editing nuclease (e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like).
  • the donor DNA template can contain sufficient homology to a genomic sequence at the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g.
  • Donor DNA template can be of any length, e.g., 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
  • a suitable donor DNA template can be from 50 nucleotides to 100 nucleotides, from 100 nucleotides to 500 nucleotides, from 500 nucleotides to 1000 nucleotides, from 1000 nucleotides to 5000 nucleotides, or from 5000 nucleotides to 10,000 nucleotides, or more than 10,000 nucleotides, in length.
  • the donor DNA template comprises a first homology arm and a second homology arm.
  • the first homology arm is at or near the 5’ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a first nucleotide sequence in a target nucleic acid.
  • the second homology arm is at or near the 3’ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a second nucleotide sequence in the target nucleic acid.
  • the first and second homology arms can each independently have a length of from about 10 nucleotides to 400 nucleotides; e.g., from 10 nucleotides (nt) to 15 nt, from 15 nt to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, from 45 nt to 50 nt, from 50 nt to 75 nt, from 75 nt to 100 nt, from 100 nt to 125 nt, from 125 nt to 150 nt, from 150 nt to 175 nt, from 175 nt to 200 nt, from 200 nt to 225 nt, from 225 nt to 250 nt, from 250 nt to 275 nt, from 275 nt to 300 nt, from 325 n
  • the donor DNA template is used for editing the target nucleotide sequence.
  • the donor DNA template comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or a combination thereof.
  • the mutation causes a shift in an open reading frame on the target polynucleotide.
  • the donor polynucleotide alters a stop codon in the target polynucleotide. In certain embodiments, the donor polynucleotide corrects a premature stop codon.
  • the correction can be achieved by deleting the stop codon, or by introducing one or more sequence changes to alter the stop codon to a codon.
  • the donor polynucleotide addresses loss of function mutations, deletions, or translocations that may occur, for example, in certain disease contexts by inserting or restoring a functional copy of a gene, or functional fragment thereof, or a functional regulatory sequence or functional fragment of a regulatory sequence.
  • a functional fragment includes a fragment less than the entire copy of a gene but otherwise provides sufficient nucleotide sequence to restore the functionality of a wild type gene or non-coding regulatory sequence (e.g., sequences encoding long non-coding RNA).
  • the donor DNA template may be used to replace a single allele of a defective gene or defective fragment thereof. In another embodiment, the donor DNA template is used to replace both alleles of a defective gene or defective gene fragment.
  • a “defective gene” or “defective gene fragment” is a gene or portion of a gene that when expressed, fails to generate a functioning protein or non-coding RNA with functionality of the corresponding wild-type gene.
  • these defective genes may be associated with one or more disease phenotypes.
  • the defective gene or gene fragment is not replaced but the heterologous nucleic acid is used to insert donor polynucleotides that encode gene or gene fragments that compensate for or override defective gene expression such that cell phenotypes associated with defective gene expression are eliminated or changed to a different or desired cellular phenotype.
  • This can be achieved by including the coding sequence of a therapeutic protein, such as a therapeutic antibody or functional fragment thereof, or a wild- type version of a defective protein associated with one or more disease phenotypes.
  • the donor may include, but not be limited to, genes or gene fragments, encoding proteins or RNA transcripts to be expressed, regulatory elements, repair templates, and the like.
  • the donor polynucleotides may comprise left end and right end sequence elements that function with transposition components that mediate insertion.
  • the donor DNA template manipulates a splicing site on the target polynucleotide.
  • the donor DNA template disrupts a splicing site. The disruption may be achieved by inserting the polynucleotide to a splicing site and/or introducing one or more mutations to the splicing site.
  • the donor polynucleotide may restore a splicing site.
  • the polynucleotide may comprise a splicing site sequence.
  • the donor DNA template to be inserted has a size from 10 bp to 50 kb in length, e.g., from 50 bp to ⁇ 40kb, from 100 bp to ⁇ 30 kb, from 100 bp to ⁇ 10 kb, from 100 bp to 300 bp, from 200 bp to 400 bp, from 300 bp to 500 bp, from 400 bp to 600 bp, from 500 bp to 700 bp, from 600 bp to 800 bp, from 700 bp to 900 bp, from 800 bp to 1000 bp, from 900 bp to 1100 bp, from 1000 bp to 1200 bp, from 1100 bp to 1300 bp, from 1200 bp to 1400 bp, from 1300 bp to 1500 bp, from 1400 bp to 1600 bp, from 1500 bp to 1700 bp,
  • the homologous arm on one or both ends of the sequence to be inserted is independently about 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, or 150 bp.
  • the first homology arm and the second homology arm of the donor DNA flank a nucleotide sequence (“a nucleotide sequence of interest” or “an intervening nucleotide sequence”) that is to be introduced into a target nucleic acid.
  • the nucleotide sequence of interest can comprise: i) a nucleotide sequence encoding a polypeptide of interest; ii) a nucleotide sequence encoding an exon of a gene; iii) a promoter sequence; iv) an enhancer sequence; v) a nucleotide sequence encoding a non-coding RNA; or vi) any combination of the foregoing.
  • the donor DNA can provide for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.
  • the donor DNA can be used to add, e.g., insert or replace, nucleic acid material to a target DNA (e.g.
  • a tag e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.
  • a regulatory sequence e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, enhancer, etc.
  • a nucleic acid sequence e.g., introduce a mutation
  • the donor DNA can be used to modify DNA in a site-specific, i.e. “targeted”, way; for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapy, e.g. to treat a disease; or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of pluripotent stem cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.
  • the donor DNA comprises a nucleotide sequence encoding a polypeptide of interest.
  • Polypeptides of interest include, e.g., a) functional versions of a polypeptide that comprises one or more amino acid substitutions, insertions, and/or deletions and that exhibits reduced function, e.g., where the reduced function is associated with or causes a pathological condition; b) fluorescent polypeptides; c) hormones; d) receptors for ligands; e) ion channels; f) neurotransmitters; g) and the like.
  • the donor DNA comprises a nucleotide sequence that encodes a wild-type protein that is lacking in the recipient cell.
  • the donor DNA encodes a wild type factor (e.g.
  • the donor DNA comprises a nucleotide sequence that encodes a therapeutic antibody.
  • the donor DNA comprises a nucleotide sequence that encodes an engineered protein or receptor.
  • the engineered receptor is a T cell receptor (TCR), a natural killer (NK) receptor (NKR), or a B cell receptor (BCR).
  • TCR T cell receptor
  • NK natural killer
  • BCR B cell receptor
  • the engineered TCR or NKR targets a cancer marker (e.g., a polypeptide that is expressed (e.g., over-expressed) on the surface of a cancer cell).
  • the donor DNA comprises a nucleotide sequence that encodes a chimeric antigen receptor (CAR).
  • CAR targets a cancer marker.
  • Donor DNAs encoding CAR, TCR, and/or NCR proteins may be folded into DNA origami structures (DNA nanostructures) and delivered into T cells or NK cells in vitro or in vivo.
  • Non-limiting examples of polypeptides that can be encoded by a donor DNA include, e.g., IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11), INS (insulin), CRP (C -reactive protein, pentraxin-related), PDGFRB (platelet- derived growth factor receptor, beta polypeptide), CCNA2 (cyclin A2), PDGFB
  • ACE angiotensin I converting enzyme peptidyl-dipeptidase A 1)
  • TNF tumor necrosis factor
  • IL6 interleukin 6 (interferon, beta 2)
  • STN statin
  • SERPINE1 serotonin peptidase inhibitor
  • clade E nonin, plasminogen activator inhibitor type 1
  • ALB albumin
  • ADIPOQ adiponectin, C1Q and collagen domain containing
  • APOB apolipoprotein B (including Ag(x) antigen)
  • APOE apolipoprotein E
  • LEP laeptin
  • MTHFR 5,10- methylenetetrahydrofolate reductase (NADPH)
  • APOA1 apolipoprotein A-I
  • EDN1 endothelin 1
  • NPPB natriuretic peptide precursor B
  • NOS3 nitric oxide synthase 3
  • GNRH1 gonadotropin-releasing hormone 1 (luteinizing- releasing hormone)
  • PAPPA pregnancy-associated plasma protein A, pappalysin 1
  • ARR3 arrestin 3, retinal (X-arrestin)
  • NPPC natriuretic peptide precursor C
  • AHSP alpha hemoglobin stabilizing protein
  • PTK2 PTK2 protein tyrosine kinase 2
  • IL13 interleukin 13
  • MTOR mechanistic target of rapamycin (serine/threonine kinase)
  • ITGB2 integratedin, beta 2 (complement component 3 receptor 3 and 4 subunit)
  • GSTT1 glutthione S-transfcrase theta 1
  • IL6ST interleukin 6 signal transducer (gpl30, oncostatin M receptor)
  • CPB2 carboxypeptidase B2 (plasma)
  • CYP1A2 cytochrome P
  • CAMP cathelicidin antimicrobial peptide
  • ZC3H12A zinc finger CCCH-type containing 12A
  • AKR1B1 aldo-keto reductase family 1, member B1 (aldose reductase)
  • DES desmin
  • MMP7 matrix metallopeptidase 7 (matrilysin, uterine)
  • AHR aryl hydrocarbon receptor
  • CSF1 colony stimulating factor 1 (macrophage)
  • HDAC9 histone deacetylase 9
  • CTGF connective tissue growth factor
  • KCNMA1 potassium large conductance calcium- activated channel, subfamily M, alpha member 1
  • UGT1A UDP glucuronosyltransferase 1 family, polypeptide A complex locus
  • PRKCA protein kinase C, alpha
  • COMT catechol-b- methyltransf erase
  • S100B S100 calcium binding protein B
  • the donor DNA encodes a wild-type version of any of the foregoing polypeptides; i.e., the donor DNA can encode a “normal” version that does not include a mutation(s) that results in reduced function, lack of function, or pathogenesis.
  • the donor DNA comprises a nucleotide sequence encoding a fluorescent polypeptide.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t- HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kae
  • fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909), and the like.
  • the donor DNA encodes an RNA, e.g., an siRNA, a microRNA, a short hairpin RNA (shRNA), an anti-sense RNA, a riboswitch, a ribozyme, an aptamer, a ribosomal RNA, a transfer RNA, and the like.
  • a donor DNA can include, in addition to a nucleotide sequence encoding one or more gene products (e.g., an RNA and/or a polypeptide), one or more transcriptional control elements, e.g., a promoter, an enhancer, and the like. In some cases, the transcriptional control element is inducible.
  • the promoter is reversible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in a eukaryotic cell. In some cases, the promoter is a cell type- specific promoter. In some cases, the promoter is a tissue-specific promoter. [00473]
  • the nucleotide sequence of the donor DNA is typically not identical to the target nucleic acid (e.g., genomic sequence) that it replaces.
  • the donor DNA may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the target nucleic acid (e.g., genomic sequence), so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease- causing base pair or a non-disease-causing base pair).
  • the donor DNA comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.
  • Donor DNA may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest (the target nucleic acid) and that are not intended for insertion into the DNA region of interest (the target nucleic acid).
  • the homologous region(s) of a donor sequence will have at least 50% sequence identity to a target nucleic acid (e.g., a genomic sequence) with which recombination is desired. In certain cases, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
  • the donor DNA may comprise certain nucleotide sequence differences as compared to the target nucleic acid (e.g., genomic sequence), where such difference include, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor DNA at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus).
  • nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein).
  • the donor DNA will include one or more nucleotide sequences to aid in localization of the donor to the nucleus of the recipient cell or to aid in the integration of the donor DNA into the target nucleic acid.
  • the donor DNA may comprise one or more nucleotide sequences encoding one or more nuclear localization signals (e.g.
  • the donor DNA will include nucleotide sequences to recruit DNA repair enzymes to increase insertion efficiency.
  • Fiuman enzymes involved in homology directed repair include MRN-CtIP, BLM-DNA2, Exol, ERCC1, Rad51, Rad52, Ligase 1, RoIQ, PARP1, Ligase 3, BRCA2, RecQ/BLM-ToroIIIa, RTEL, Ro ⁇ d, and Ro ⁇ h (Verma and Greenburg (2016) Genes Dev.30 (10): 1138-1154).
  • the donor DNA is delivered as reconstituted chromatin (Cruz-Becerra and Kadonaga (2020) eLife 2020;9:e55780 DOI: 10.7554/eLife.55780).
  • the ends of the donor DNA are protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art.
  • one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or self complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.
  • the donor / template comprises a coding sequence for a functional RNA element (ncRNAs, siRNA, shRNA, sgRNA, etc.).
  • the heterologous nucleic acid sequences encode a functional non-translated RNA.
  • the functional non-translated RNA is an RNA aptamer or a ribozyme.
  • the heterologous nucleic acid of the engineered retrons further includes a unique barcode to facilitate multiplexing. Barcodes may include one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Such barcodes may be inserted for example, into the tip/loop region of the msd- encoded DNA.
  • Barcodes can be 3-1000 or more nucleotides in length, 10-250 nucleotides in length, or 10-30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length.
  • barcodes are also used to identify the position (viz., positional barcode) of a cell, colony, or sample from which a retron originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, the position of a tube in a rack, or the location of a sample in a laboratory.
  • a barcode may be used to identify the position of a genetically modified cell containing a retron. The use of barcodes allows retrons from different cells to be pooled in a single reaction mixture for sequencing while still being able to trace a particular retron back to the colony from which it originated.
  • adapter sequences can be added to engineered retron to facilitate high-throughput amplification or sequencing.
  • a pair of adapter sequences can be added at the 5 ⁇ and 3 ⁇ ends of a retron construct to allow amplification or sequencing of multiple engineered retron simultaneously by the same set of primers.
  • HNS guide RNA
  • the functional non-translated RNA is a CRISPR/Cas guide RNA (gRNA) specific for a target sequence in the mammalian cell.
  • FIG.1G depicts various configurations of a recombinant retron ncRNA which is modified by inserting a guide RNA at the 5’ end or the 3’ of the ncRNA.
  • the guide RNA also may be provided in trans as a separate construct. In addition, guide RNAs may be placed at both ends of a recombinant retron ncRNA. [00482] The skilled person will understand that selection of an appropriate guide RNA is informed by which RNA-guided nuclease is utilized. [00483]
  • a guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.
  • guide RNA refers to an RNA that comprises: i) an “activator” nucleotide sequence that binds to a CRISPR/Cas effector polypeptide (e.g., a class 2 CRISPR/Cas effector polypeptide such as a type II, type V, or type VI CRISPR/Cas endonuclease) and activates the CRISPR/Cas effector polypeptide; and ii) a “targeter” nucleotide sequence that comprises a nucleotide sequence that hybridizes with a target nucleic acid.
  • a CRISPR/Cas effector polypeptide e.g., a class 2 CRISPR/Cas effector polypeptide such as a type II, type V, or type VI CRISPR/Cas endonuclease
  • the “activator” nucleotide sequence and the “targeter” nucleotide sequence can be on separate RNA molecules (e.g., a “dual-guide RNA”); or can be on the same RNA molecule (a “single-guide RNA”).
  • a guide nucleic acid in some cases includes only ribonucleotides. In some cases, a guide nucleic acid includes both ribonucleotides and deoxyribonucleotides.
  • a CRISPR/Cas guide RNA comprises one or more modifications, e.g., a base modification, a backbone modification, a sugar modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability, such as improved in vivo stability).
  • modifications e.g., a base modification, a backbone modification, a sugar modification, etc.
  • Suitable nucleic acid modifications include, but are not limited to: 2’O-methyI modified nucleotides, 2’ fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7-methyIguanyIate cap (m7G)).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • Suitable modified nucleic acid backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-aIkyIene phosphonates, 5'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates 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
  • Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium
  • mixed salts and free acid forms are also included.
  • a CRISPR- Cas guide RNA can also include one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl.
  • Particularly suitable are 0((CH2)n0) mCH3, 0(CH2)n0CH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)n0NH2, and 0(CH 2 ) n 0N((CH 2 ) n CH 3 ) 2 , where n and m are from 1 to about 10.
  • Suitable polynucleotides comprise a sugar substituent group selected from: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , 0N0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a sugar substituent group selected from: Ci to Cio lower alkyl,
  • a suitable modification includes 2'-methoxy ethoxy (2'-0-CH2 CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'- MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'- DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'- DMAEOE), i.e., 2'-0-CH 2 -0-CH 2 -N(CH 3 ) 2 .
  • Examples of various CRISPR/Cas effector proteins and CRISPR/Cas guide RNAs can be found in the art, for example, see Jinek et al., Science.2012 Aug 17;337(6096):816-21 ; Chylinski et al., RNA Biol.2013 May;10(5):726-37; Ma et al., Biomed Res Int.2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15644-9; Jinek et al., Elife.2013;2:e00471; Pattanayak et al., Nat Biotechnol.2013 Sep;31(9):839-43; Qi et al., Cell.2013 Feb 28 ; 152(5): 1173-83; Wang ek et al., Science.2012 Aug 17;337(6096):816-21 ; Chylinski et
  • Reverse Transcriptases [00487] Reverse transcriptases (RTs, also known as RNA-directed DNA polymerases) are enzymes present in all three domains of life, which are DNA polymerase using RNA as a template. Reverse transcriptases of the present disclosure are used to reverse transcribe template msd RNA into single-stranded msDNA. [00488]
  • the reverse transcriptase or a functional domain thereof that may be used in the instant invention includes prokaryotic and eukaryotic RT, provided that the RT functions within the host to generate a donor polynucleotide sequence from the RNA template (e.g., an RNA template from the retron transcript ncRNA).
  • suitable RT sequences are provided in Table A.
  • nucleotide sequence of a native or wild-type RT is modified, for example, using known codon optimization techniques, so that expression within the desired host is optimized.
  • the RT domain of a reverse transcriptase is used in the present invention, so long as it is compatible with the engineered retron of the invention. The domain may include only the RNA-dependent DNA polymerase activity.
  • the RT domain is non-mutagenic, i.e., does not cause mutation in the donor polynucleotide (e.g., during the reverse transcriptase process).
  • the RT domain may be non-retron RT in origin, e.g., a viral RT or a human endogenous RTs.
  • the RT domain is retron RT or DGRs RT.
  • the RT may be less mutagenic than a counterpart wildtype RT. In certain embodiments, the RT is not mutagenic.
  • a reverse transcriptase is encoded by a retron ret gene, which may accompany the cognate msr and msd loci and specifically recognize the secondary structure of the cognate ncRNA transcript.
  • the RT may be obtained from prokaryotic or eukaryotic cells. Most reverse transcriptases (80%) can be phylogenetically clustered into three major lineages: group II introns, diversity-generating retroelements (DGRs), and retrons.
  • RTs include abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II-like (G2L), the unknown groups (UG), and rvt elements.
  • Abi abortive infection
  • CRISPR-Cas-associated RTs Group II-like (G2L), the unknown groups (UG), and rvt elements.
  • the RT gene is a cognate RT, a retron RT from a species within the same species or clade of the cognate RT, or a retron RT not within the same clade of the cognate RT such as an unrelated RT or an engineered RT.
  • the non- retron related RT are RTs from group II introns, diversity-generating retroelements (DGRs), abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II-like (G2L), the unknown groups (UG), and rvt elements.
  • DGRs diversity-generating retroelements
  • Abi abortive infection
  • CRISPR-Cas-associated RTs Group II-like (G2L)
  • G2L Group II-like
  • UG unknown groups
  • the RT are from clades related to retron/retron-like sequences. In some embodiments, the RT are selected from RTs provided in Table A. In some embodiments, the RT is not an RT associated with the sequences identified in Table X. [00496] In prokaryotic retron systems, the RT gene is typically located downstream from the ncRNA (msr and msd) locus. In the engineered retron, the RT position can differ from the natural or wild type retrons. In some embodiments, the RT gene can be provided in cis, such as either upstream or downstream of the msr locus or the msd locus.
  • the RT gene is provided in trans, such as provided separately in a vector of the vector system described herein, wherein the ncRNA coding msr and msd sequences are provided in a different vector of the vector system described herein.
  • the RT is modified (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) or codon optimized to enhance activity or processivity.
  • a cryptic stop signal is removed from the RT thereby allowing generation of longer ssDNAs.
  • the RT is from a retron which encodes msDNA as described in US 6,017,737; US5,849,563; US5,780,269; US5,436,141; US5,405,775; US5,320,958; CA2,075,515; all of which are incorporated by reference herein in their entireties.
  • the engineered retron further comprises a polynucleotide (e.g., a DNA molecule) encoding a reverse transcriptase (RT) or a portion thereof.
  • the encoded RT or portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.
  • the polynucleotide (e.g., a DNA molecule) encoding the RT comprises a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A, or
  • the polynucleotide encoding the RT encodes a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or a polypeptide of Table C.
  • the polynucleotide encoding the RT does not comprise a polynucleotide listed in Table X.
  • the RT binds the ncRNA template downstream from the msd locus, forming an RT-RNA complex, and initiating reverse transcription of the RNA towards its 5 ⁇ end. Accordingly, in certain aspects the disclosure relates to an engineered nucleic acid- enzyme construct comprising: a.
  • ncRNA non-coding RNA
  • msDNA multi-copy single-stranded DNA
  • msd locus encoding the msd RNA portion of the msDNA
  • a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c.
  • a reverse transcriptase or a domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C.
  • the RT does not comprise a polypeptide listed in Table X.
  • the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA, b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT), or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the
  • the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in Table A, or an RT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
  • the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and/or optionally wherein the ncRNA is not an ncRNA from the retons of Table X.
  • the RT is linked to components such as RNA-guided and non-RNA guided nucleases.
  • the linked maybe via s peptide bond or a short linker peptide in a fusion protein.
  • Suitable linker peptides include flexible linkers such as those comprising G or S repeats, such as G 4 S repeat units or GS repeat units, with 1-20 repeats, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 repeats.
  • the RT is chemically linked or conjugated to the RNA- guided and non-RNA guided nucleases via non-peptide bonds.
  • Such protein conjugates may be delivered directly to a host cell, either together with the nucleic acid component of the engineered retron described herein, or separately.
  • the RT is linked to a DNA-repair modulating biomolecule (e.g., NHEJ peptide inhibitors.
  • RNA-guided Nucleases RNA-guided Nucleases
  • the engineered retron e.g., an engineered nucleic acid construct or engineered nucleic acid-enzyme construct as described herein
  • gRNA guide RNA
  • the gRNA includes sequences complementary to a genomic sequence, and therefore can mediate binding of the nuclease-gRNA complex to the genomic target site by hybridization between the guide sequence and the target site sequence.
  • the gRNA can be linked to the ncRNA and/or the msDNA encoded by the engineered retron described herein at the 5 ⁇ end of the ncRNA and/or the msDNA.
  • the gRNA can be linked to the ncRNA and/or the msDNA encoded by the engineered retron described herein at the 3 ⁇ end of the RNA of the ncRNA and/or the msDNA after reverse transcription.
  • the nuclease that can form a complex with the gRNA can be any one of the art-recognized clustered regularly interspersed short palindromic repeats (CRISPR) system Cas effector enzymes, which are useful for, e.g., genome editing, including genome editing in mammalian cells or human cells.
  • CRISPR CRISPR
  • a gRNA that can be loaded into a Cas9 or variant thereof may be encoded by the engineered retron, such that the gRNA is transcribed as part of the msDNA.
  • the gRNA may be linked to the 5 ⁇ end of the a1 region of the msr-encoded sequence in the retron ncRNA, as well as the msDNA after reverse transcription.
  • the gRNA may be part of the modified msr-region present in the ncRNA and the msDNA produced after reverse transcription (viz., the encoded gRNA is not degraded by the synthesis of the msDNA through reverse transcription of the ncRNA).
  • Cas enzymes Any art recognized CRISPR/Cas effector enzymes or variants thereof (“Cas enzymes”) known to be useful for CRISPR-based genome editing can be used with the engineered retron, though such Cas enzymes may not necessarily be part of the engineered retron, and can (but is not required to) be provided separately.
  • the Cas enzymes can be provided as part of the vector system described herein.
  • the coding sequence of the suitable Cas enzyme can be present on the same vector that provides the engineered retron, or on different vectors.
  • the engineered retron and the Cas enzymes When the engineered retron and the Cas enzymes are present on the same vector, they may be under the transcriptional control of the same promoter, enhancer, or other transcriptional regulatory elements, or may be separately regulated by different promoters, enhancers, and/or other transcriptional regulatory sequences in the vector system.
  • the Cas enzyme is a Class 2, Type II CRISPR/Cas effector enzyme, such as Cas9.
  • the Cas9 is from Streptococcus pyogenes (SpCas9)
  • the gRNA encoded by the engineered retron comprises both the crisprRNA (crRNA) and the tracrRNA linked together as the single guide RNA (gRNA).
  • the Cas9 is from Staphylococcus aureus Cas9 (SaCas9), or an engineered variant thereof such as SaCas9-HF (a high-fidelity variant with genome-wide activity), KKHSaCas9 (which recognizes a 5 ⁇ -NNGRRT-3 ⁇ PAM, and has a 2-4x broader range of target sites in the human genome than the wildtype SaCas9), and microABE1744 (an engineered SaCas9 variant adapted for use in adenine base editing (ABE), with significantly improved on-target editing compared to other nucleases, with a reduced RNA off-target footprint).
  • SaCas9-HF a high-fidelity variant with genome-wide activity
  • KKHSaCas9 which recognizes a 5 ⁇ -NNGRRT-3 ⁇ PAM, and has a 2-4x broader range of target sites in the human genome than the wildtype SaCas9
  • microABE1744 an engineered SaCas9 variant adapted for
  • the Cas9 is from Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), or Campylobacter jejuni (CjCas9).
  • the Cas9 is from Streptococcus canis (ScCas9), with a less stringent PAM sequence requirement of 5′-NNG-3′ (instead of the more stringent 5′-NGG-3′ for SpCas9).
  • the Cas9 is from Staphylococcus auricularis (SauriCas9) (which recognizes a 5 ⁇ -NNGG-3 ⁇ PAM sequence, has high editing activity).
  • the Cas enzyme is a Cas9 variant with mutated catalytic domain that retains cleavage specificity, but only nicks a single DNA strand at a desired target sequence instead of creating a double-strand break (DSB). Two such Cas9 nickase variants targeting different strands of the same target sequence may be used together to increase fidelity of creating the DSB, each using a different gRNA that can be provided by the engineered retron.
  • the Cas enzyme is a high fidelity Cas9 variant with weakened DNA phosphate backbone interaction (such as SpCas9-HF1) that displays genome- wide specificity and undetectable off-target effects.
  • the Cas enzyme is a Cas9 variant known as eSpCas9, which has weakened interactions between the eSpCas9 and its gRNAs with non-exact complementarity with the target DNA sequence, thus providing improved specificity and lower off-target editing rates.
  • the Cas enzyme is a hyper-accurate Cas9 variant (HypaCas9), which improves proofreading before cleavage, and thus drastically reduces off- target cleavage.
  • the Cas enzyme is a Cas9 variant (FokI-Fused dCas9), which combines the DNA recognition ability by dCas9 with the specificity of an active nuclease FokI. The resulting nuclease cuts the target sequence only after dimerization, which is more difficult to occur at off-target sites, thus resulting in enhanced specificity.
  • the Cas enzyme is a Cas9 variant xCas9, which recognizes a broad range of PAM sequences, thus increasing the target sites to 1 in 4 in the genome.
  • the xCas9 variant also exhibits lower off-target rates than the commonly used SpCas9.
  • the Cas enzyme is a Cas9 variant with altered PAM sequence specificities, including the SpG variant with an expanded target range of PAM sequences, and the SpRY variant that can target almost all PAM sequences.
  • the Cas enzyme is dCas9 having inactivated catalytic nuclease domains while maintaining the recognition domains that allow guide RNA-mediated targeting to specific DNA sequences.
  • the dCas9 may be further linked to a functional domain with a distinct biological function, such as transcriptional activation / depression, DNA methylation, demethylation, endonuclease (such as FokI), or fluorescent dye.
  • Representative (non-limiting) dCas9-linked functional domains include including dCas9-SAM, dCas9-SunTag, dCas9-VPR, and dCas9-KREB.
  • the dCas9 is fused with a catalytic enzyme with cytidine deaminase activity, which converts GC basepair to AT basepair.
  • the dCas9 is fused with an engineered RNA adenosine deaminase, which converts AT basepair to GC basepair.
  • the Cas enzyme is a Class 2, Type V CRISPR/Cas effector enzyme, such as Cas12a (Cpf1) (Type V-A), Cas12b (C2c1) (Type V-B), Cas12c (C2c3) (Type V-C), Cas12d (CasY) (Type V-D), Cas12e (CasX) (Type V-E), Cas12f (Cas14, C2c10) (Type V- F), Cas12g (Type V-G), Cas12h (Type V-H), Cas12i (Type V-I), Cas12k (C2c5) (Type V-K), or C2c4 / C2c8 / C2c9 (Type V-U).
  • the Cas enzyme is Cas12a or Cpf1 (CRISPR from Prevotella and Francisella 1). Unlike Cas9, Cas12a is well-suited for targeting AT-rich DNA sequences because of its AT-rich PAM sequences.
  • the Cas12a is FnCas12a (that recognizes PAM sequence 5′ ⁇ TTN ⁇ 3′), or AsCas12a or LbCas12a (that recognize 5′ ⁇ TTTV ⁇ 3′ PAM sequence), where V is A, G, or C nucleotide.
  • Cas12a creates staggered double-stranded breaks in the target DNA, rather than the blunt-ends generated by SpCas9, thus rendering it more useful for HDR repair.
  • the subject engineered retron encodes a gRNA suitable for use for Cas12a, in that the gRNA does not require a tracer RNA, and only requires the crRNA.
  • the Cas enzyme is a Cas12a variant from Acidaminococcus sp. (enAsCas12a), which has an expanded target range of PAM sequences and significantly higher editing activity compared to wild type Cas12a.
  • the Cas enzyme is a high-fidelity Cas12a variant (enAsCas12a-HF1) that reduces off-target editing.
  • the Cas enzyme is Cas12b or C2c1.
  • the Cas12b is form Alicyclobacillus acidoterrestris (AacCas12b), or from Alicyclobacillus acidiphilus Cas12b (AapCas12b).
  • the Cas enzyme is a Cas12b variant that functions at 37°C, such as one form Bacillus hisashii (BhCas12b).
  • the Cas enzyme is a BhCas12b variant with higher specificity than SpCas9. [00538] In some embodiments, the Cas enzyme is CasX or Cas12d. [00539] In some embodiments, the Cas enzyme is CasY or Cas12e, and the subject engineered retron encodes a short-complementarity untranslated RNA (scoutRNA) together with crRNA (rather than tracrRNA used in other CRISPR-Cas systems). [00540] In some embodiments, the Cas enzyme is Cas14, from archaea bacteria.
  • Cas14 targets single-stranded (ss) DNA target sequences, does not require PAM sequence for activation, and has collateral activity (i.e., cuts other non-target ssDNA strands non-specifically upon binding the target sequence). Unlike Cas12a, Cas14a requires high fidelity complementarity to the target ssDNA, and is very sensitive to internal seed region mismatches of the ssDNA target substrate.
  • the gRNA nuclease is an engineered RNA-guided Fokl nuclease.
  • RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA- dependent targeting on Fokl.
  • dCas9 inactive Cas9
  • FokI-dCas9 Fokl endonuclease
  • dCas9 portion confers guide RNA- dependent targeting on Fokl.
  • the RNA-guided nuclease can be a non-CRISPER/Cas related nuclease such as transposon-encoded nucleases, IscBs, IscR, or TnpBs.
  • the Cas enzyme is a Cas9
  • the retron-based editing systems described herein can include any Cas9 equivalent.
  • a Cas9 equivalent is a Cas9-like protein that provides the same or substantially the same function as Cas9.
  • Cas9 refers to a type II enzyme of the CRISPR-Cas system
  • a Cas9 equivalent can refer to a type V or type VI enzyme of the CRISPR- Cas system.
  • Cas12e is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution.
  • the Cas12e (CasX) protein is contemplated to be used with the retron-based editor systems described herein.
  • any variant or modification of Cas12e (CasX) is conceivable and within the scope of the present disclosure.
  • Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), which have been described in, for example, Burstein et al.,“New CRISPR–Cas systems from uncultivated microbes.” Cell Res.2017 Feb 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference.
  • CasX Cas12e
  • CasY Cas12d
  • Cas9 refers to Cas12e, or a variant of Cas12e. In some embodiments, Cas9 refers to a Cas12d, or a variant of Cas12d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.
  • napDNAbp nucleic acid programmable DNA binding protein
  • the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein.
  • the napDNAbp is a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein.
  • the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.
  • the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1.
  • Cas9 e.g., dCas9 and nCas9
  • Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9.
  • Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break.
  • Cpf1- family proteins Two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells.
  • Cpf1 proteins are known in the art and have been described previously, for example Yamano et al.,“Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference.
  • the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
  • CRISPR associated protein including but not limited to, Cas12a, Cas12b
  • the RNA-guided nuclease can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, or an Argonaute (Ago) domain, or a variant thereof.
  • a Cas9 a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1
  • RNA-guided nucleases are readily available and known in the art. Exemplary RNA-guided nucleases and their amino acid sequences can be found, for example, in WO 2017/070633, US 2020/0010835, US 2022/0204975, US 11071790, WO 2020/191233, US 11447770, US 10858639, and US 10947530, each of which are incorporated herein by reference in their entireties. E.
  • the subject engineered retron can also be used in combination with sequence-specific nucleases that do not use a guide RNA to recognize a target sequence, such as non-CRISPR/Cas sequence-specific nucleases including TALENs, ZFNs, meganucleases, and restriction enzymes, as well as other sequence-specific nucleases that use other RNA guides, such as transposon-encoded IscBs, IscR, or TnpBs.
  • the subject engineered retron may encode or provide a msDNA that can serve as a donor or template sequence for HDR-mediated genome editing.
  • the RT of the engineered retron is fused to such sequence-specific nuclease, such that the msDNA, by way of being generated by the RT close to the site of HDR-mediated genome editing, can be more efficiently participate in the HDR-mediated genome editing.
  • the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE Nuclease, a TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, meganuclease, or a combination thereof.
  • the non-CRISPR/Cas sequence- specific nuclease is or includes two, three, four, or more of an independently selected TALE Nuclease, TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, Meganuclease, restriction enzymes or a combination thereof.
  • the combination is or comprises a TALE Nuclease/a ZF Nuclease; a TALE Nickase/a ZF nickase.
  • the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE Nuclease (Transcription Activator-Like Effector Nucleases (TALEN)).
  • TALE Nuclease Transcription Activator-Like Effector Nucleases (TALEN)
  • TALENs are restriction enzymes engineered to cut specific target DNA sequences.
  • TALENs comprise a TAL effector (TALE) DNA-binding domain (which binds at or close to the target DNA), fused to a DNA cleavage domain which cuts target DNA.
  • TALEs are engineered to bind to practically any desired DNA sequence.
  • the TALEN comprises an N-terminal capping region, a DNA binding domain which may comprise at least one or more TALE monomers or half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein these three parts are arranged in a predetermined N- terminus to C-terminus orientation.
  • the TALEN includes at least one or more regulatory or functional protein domains.
  • the TALE monomers or half monomers may be variant TALE monomers derived from natural or wild type TALE monomers but with altered amino acids at positions usually highly conserved in nature, and in particular have a combination of amino acids as RVDs that do not occur in nature, and which may recognize a nucleotide with a higher activity, specificity, and/or affinity than a naturally occurring RVD.
  • the variants may include deletions, insertions and substitutions at the amino acid level, and transversions, transitions and inversions at the nucleic acid level at one or more locations.
  • the variants may also include truncations.
  • the TALE monomer / half monomer variants include homologous and functional derivatives of the parent molecules.
  • the variants are encoded by polynucleotides capable of hybridizing under high stringency conditions to the parent molecule-encoding wild-type nucleotide sequences.
  • the DNA binding domain of the TALE has at least 5 of more TALE monomers and at least one or more half-monomers specifically ordered or arranged to target a genomic locus of interest.
  • the construction and generation of TALEs or polypeptides of the invention may involve any of the methods known in the art.
  • Naturally occurring TALEs or “wild type TALEs” are nucleic acid binding proteins secreted by numerous species of proteobacteria.
  • TALEs contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides that are predominantly 33, 34 or 35 amino acids in length and that differ from each other mainly in amino acid positions 12 and 13.
  • a general representation of a TALE monomer which is comprised within the DNA binding domain is Xl-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid.
  • X12X13 indicate the RVDs.
  • the variable amino acid at position 13 is missing or absent and in such monomers, the RVD consists of a single amino acid.
  • the RVD may be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent.
  • the DNA binding domain may comprise several repeats of TALE monomers and this may be represented as (Xl-11-(X12X13)-X14-33 or 34 or 35)z, where z is optionally at least 5- 40, such as 10-26.
  • the TALE monomers have a nucleotide binding affinity that is determined by the identity of the amino acids in its RVD.
  • Polypeptide monomers with an RVD of NI preferentially bind to adenine (A), monomers with an RVD of NG preferentially bind to thymine (T), monomers with an RVD of HD preferentially bind to cytosine (C), monomers with an RVD of NN preferentially bind to both adenine (A) and guanine (G), monomers with an RVD of IG preferentially bind to T, monomers with an RVD of NS recognize all four base pairs and may bind to A, T, G or C.
  • the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity.
  • TALEs The structure and function of TALEs is further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety.
  • the TALE is a dTALE (or designerTALE), see Zhang et al., Nature Biotechnology 29:149-153 (2011), incorporated herein by reference.
  • the TALE monomer comprises an RVD of HN or NH that preferentially binds to guanine, and the TALEs have high binding specificity for guanine containing target nucleic acid sequences.
  • polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS preferentially bind to guanine.
  • polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN preferentially bind to guanine.
  • polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind to guanine.
  • the RVDs that have high binding specificity for guanine are RN, NH RH and KH.
  • polypeptide monomers having an RVD of NV preferentially bind to adenine and guanine as do monomers having the RVD HN.
  • Monomers having an RVD of NC preferentially bind to adenine, guanine and cytosine, and monomers having an RVD of S (or S*), bind to adenine, guanine, cytosine and thymine with comparable affinity.
  • monomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thymine with comparable affinity.
  • Such polypeptide monomers allow for the generation of degenerative TALEs able to bind to a repertoire of related, but not identical, target nucleic acid sequences.
  • the TALE polypeptide has a nucleic acid binding domain containing polypeptide monomers arranged in a predetermined N-terminus to C-terminus order such that each polypeptide monomer binds to a nucleotide of a predetermined target nucleic acid sequence, and where at least one of the polypeptide monomers has an RVD of HN or NH and preferentially binds to guanine, an RVD of NV and preferentially binds to adenine and guanine, an RVD of NC and preferentially binds to adenine, guanine and cytosine or an RVD of S and binds to adenine, guanine, cytosine and thymine.
  • each polypeptide monomer of the nucleic acid binding domain that binds to adenine has an RVD of NI, NN, NV, NC or S.
  • each polypeptide monomer of the nucleic acid binding domain that binds to guanine has an RVD of HN, NH, NN, NV, NC or S.
  • each polypeptide monomer of the nucleic acid binding domain that binds to cytosine has an RVD of HD, NC or S.
  • each polypeptide monomer that binds to thymine has an RVD of NG or S.
  • each polypeptide monomer of the nucleic acid binding domain that binds to adenine has an RVD of NI.
  • each polypeptide monomer of the nucleic acid binding domain that binds to guanine has an RVD of HN or NH.
  • each polypeptide monomer of the nucleic acid binding domain that binds to cytosine has an RVD of HD.
  • each polypeptide monomer that binds to thymine has an RVD of NG.
  • the RVDs that have a specificity for adenine are NI, RI, KI, HI, and SI. [00573] In certain embodiments, the RVDs that have a specificity for adenine are HN, SI and RI, most preferably the RVD for adenine specificity is SI. [00574] In certain embodiments, the RVDs that have a specificity for thymine are NG, HG, RG and KG. [00575] In certain embodiments, the RVDs that have a specificity for thymine are KG, HG and RG, most preferably the RVD for thymine specificity is KG or RG.
  • the RVDs that have a specificity for cytosine are HD, ND, KD, RD, HH, YG and SD.
  • the RVDs that have a specificity for cytosine are SD and RD.
  • FIG.4B of WO 2012/067428 provides representative RVDs and the nucleotides they target, the entire content of which is hereby incorporated herein by reference.
  • the variant TALE monomers may comprise any of the RVDs that exhibit specificity for a nucleotide as depicted in FIG.4A of WO2012/067428.
  • the RVD SH may have a specificity for G
  • the RVD IS may have a specificity for A
  • the RVD IG may have a specificity for T.
  • the RVD NT may bind to G and A.
  • the RVD NP may bind to A, T and C.
  • At least one selected RVD may be NI, HD, NG, NN, KN, RN, NH, NQ, SS, SN, NK, KH, RH, HH, KI, HI, RI, SI, KG, HG, RG, SD, ND, KD, RD, YG, HN, NV, NS, HA, S*, N*, KA, H*, RA, NA or NC.
  • the predetermined N-terminal to C-terminal order of the one or more polypeptide monomers of the nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the TALE or polypeptides of the invention may bind.
  • the monomers and at least one or more half monomers are “specifically ordered to target” the genomic locus or gene of interest.
  • the natural TALE-binding sites always begin with a thymine (T), which may be specified by a cryptic signal within the non-repetitive N-terminus of the TALE polypeptide; in some cases this region may be referred to as repeat 0.
  • TALE binding sites do not necessarily have to begin with a thymine (T) and polypeptides of the invention may target DNA sequences that begin with T, A, G or C.
  • tandem repeat of TALE monomers always ends with a half- length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full length TALE monomer and this half repeat may be referred to as a half- monomer (FIG.8 of WO 2012/067428). Therefore, it follows that the length of the nucleic acid or DNA being targeted is equal to the number of full monomers plus two (see FIG.44 of WO 2012/067428).
  • nucleic acid binding domains are engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more polypeptide monomers arranged in a N-terminal to C-terminal direction to bind to a predetermined 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotide length nucleic acid sequence.
  • nucleic acid binding domains are engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more full length polypeptide monomers that are specifically ordered or arranged to target nucleic acid sequences of length 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 nucleotides, respectively.
  • the polypeptide monomers are contiguous.
  • half-monomers may be used in the place of one or more monomers, particularly if they are present at the C-terminus of the TALE.
  • Polypeptide monomers are generally 33, 34 or 35 amino acids in length.
  • the amino acid sequences of polypeptide monomers are highly conserved or as described herein, the amino acids in a polypeptide monomer, with the exception of the RVD, exhibit patterns that effect TALE activity, the identification of which may be used in preferred embodiments of the invention.
  • the DNA binding domain may comprise (Xl-11- X12X13-X14-33 or 34 or 35)z, wherein Xl-11 is a chain of 11 contiguous amino acids, wherein X12X13 is a repeat variable diresidue (RVD), wherein X14-33 or 34 or 35 is a chain of 21, 22 or 23 contiguous amino acids, wherein z is at least 5 to 26, then the preferred combinations of amino acids are LTLD (SEQ ID NO:19936) or LTLA (SEQ ID NO:19937) or LTQV (SEQ ID NO:19938) at Xl-4, or EQHG (SEQ ID NO:19939) or RDHG (SEQ ID NO:19940) at positions X30-33 or X31-34 or X32-35.
  • RVD repeat variable diresidue
  • LTPD SEQ ID NO:19941
  • NQALE SEQ ID NO:19942
  • DHG DHG
  • LTPD SEQ ID NO:19941
  • NQALE SEQ ID NO:19942
  • DHG DHG at X32-34 when the monomer is 34 amino acids in length.
  • NQALE SEQ ID NO:19942
  • DHG DHG is at X31-33 or X33-35.
  • amino acid combinations of interest in the monomers are LTPD at Xl-4 and KRALE (SEQ ID NO:19943) at X16-20 and AHG at X32-34 or LTPE (SEQ ID NO:19944) at Xl-4 and KRALE (SEQ ID NO:19943) at XI 6-20 and DHG at X32-34 when the monomer is 34 amino acids in length.
  • the monomer is 33 or 35 amino acids long, the corresponding shift occurs in the positions of the contiguous amino acids KRALE (SEQ ID NO:19943), AHG and DHG.
  • the positions of the contiguous amino acids may be (LTPD at Xl-4 and KRALE (SEQ ID NO:19943) at X15-19 and AHG at X31-33) or (LTPE at Xl-4 and KRALE (SEQ ID NO:19943) at X15-19 and DHG at X31-33) or (LTPD at Xl-4 and KRALE at X17-21 and AHG at X33-35) or (LTPE at Xl-4 and KRALE (SEQ ID NO:19943) at X17-21 and DHG at X33-35).
  • contiguous amino acids [NGKQALE] (SEQ ID NO:19945) are present at positions X14-20 or X13-19 or X15-21. These representative positions put forward various embodiments of the invention and provide guidance to identify additional amino acids of interest or combinations of amino acids of interest in all the TALE monomers (see FIGs.24A-24F, and 25 of WO 2012/067428).
  • Exemplary amino acid sequences of conserved portions of polypeptide monomers are provided below. The position of the RVD in each sequence is represented by XX or by X* (wherein (*) indicates that the RVD is a single amino acid and residue 13 (X13) is absent).
  • TALE polypeptide binding efficiency is increased by including amino acid sequences from the “capping regions” that are directly N-terminal or C- terminal of the DNA binding region of naturally occurring TALEs into the engineered TALEs at positions N-terminal or C-terminal of the engineered TALE DNA binding region.
  • the TALE polypeptides described herein further comprise an N-terminal capping region and/or a C-terminal capping region.
  • An exemplary amino acid sequence of a N-terminal capping region is:
  • An exemplary amino acid sequence of a C-terminal capping region is: [00595]
  • the DNA binding domain comprising the repeat TALE monomers and the C-terminal capping region provide structural basis for the organization of different domains in the d-TALEs or polypeptides of the invention.
  • N-terminal and/or C-terminal capping regions are not necessary to enhance the binding activity of the DNA binding region. Therefore, in certain embodiments, fragments of the N-terminal and/or C-terminal capping regions are included in the TALE polypeptides described herein.
  • the TALE (including TALEs) polypeptides described herein contain a N-terminal capping region fragment that included at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino acids of an N-terminal capping region.
  • the N-terminal capping region fragment amino acids are of the C-terminus (the DNA-binding region proximal end) of an N-terminal capping region.
  • N-terminal capping region fragments that include the C-terminal 240 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 147 amino acids retain greater than 80% of the efficacy of the full length capping region, and fragments that include the C-terminal 117 amino acids retain greater than 50% of the activity of the full-length capping region.
  • the TALE polypeptides described herein contain a C- terminal capping region fragment that included at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, 180 amino acids of a C-terminal capping region.
  • the C-terminal capping region fragment amino acids are of the N-terminus (the DNA-binding region proximal end) of a C-terminal capping region.
  • C-terminal capping region fragments that include the C-terminal 68 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 20 amino acids retain greater than 50% of the efficacy of the full length capping region.
  • the capping regions of the TALE polypeptides described herein do not need to have identical sequences to the capping region sequences provided herein.
  • the capping region of the TALE polypeptides described herein have sequences that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or share identity to the capping region amino acid sequences provided herein.
  • Sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • the capping region of the TALE polypeptides described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.
  • Sequence homologies may be generated by any of a number of computer programs known in the art, which include but are not limited to BLAST or FASTA. Suitable computer program for carrying out alignments like the GCG Wisconsin Bestfit package may also be used. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
  • % homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • the TALEs described herein also include a nuclear localization signal and/or cellular uptake signal. Such signals are known in the art and may target a TALE to the nucleus and/or intracellular compartment of a cell. Such cellular uptake signals include, but are not limited to, the minimal Tat protein transduction domain which spans residues 47-57 of the human immunodeficiency virus Tat protein: YGRKKRRQRRR (SEQ ID NO:19952). [00603] In some embodiments, the TALEs described herein include a nucleic acid or DNA binding domain that is a non-TALE nucleic acid or a non-TALE DNA binding domain.
  • non-TALE DNA binding domain refers to a DNA binding domain that has a nucleic acid sequence corresponding to a nucleic acid sequence which is not substantially homologous to a nucleic acid that encodes for a TALE protein or fragment thereof, e.g., a nucleic acid sequence which is different from a nucleic acid that encodes for a TALE protein and which is derived from the same or a different organism.
  • the TALEs described herein include a nucleic acid or DNA binding domain that is linked to a non-TALE polypeptide.
  • a “non-TALE polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a TALE protein or fragment thereof, e.g., a protein which is different from a TALE protein and which is derived from the same or a different organism.
  • the term “linked” is intended include any manner by which the nucleic acid binding domain and the non-TALE polypeptide could be connected to each other, including, for example, through peptide bonds by being part of the same polypeptide chain or through other covalent interactions, such as a chemical linker.
  • the non- TALE polypeptide may be linked, for example to the N-terminus and/or C-terminus of the nucleic acid binding domain, may be linked to a C-terminal or N-terminal cap region, or may be connected to the nucleic acid binding domain indirectly.
  • the TALEs or polypeptides of the invention comprise chimeric DNA binding domains.
  • Chimeric DNA binding domains may be generated by fusing a full TALE (including the N- and C- terminal capping regions) with another TALE or non-TALE DNA binding domain such as zinc finger (ZF), helix-loop-helix, or catalytically-inactivated DNA endonucleases (e.g., EcoRI, meganucleases, etc.), or parts of TALE may be fused to other DNA binding domains.
  • the chimeric domain may have novel DNA binding specificity that combines the specificity of both domains.
  • the TALE polypeptides of the invention include a nucleic acid binding domain linked to the one or more effector domains.
  • the effector domain is a nickase or nuclease.
  • the sequence-specific nuclease is a zinc finger nuclease (ZFN), such as an artificial zinc-finger nuclease having arrays of zinc-finger (ZF) modules to target new DNA-binding sites in a target sequence (e.g., target sequence or target site in the genome).
  • ZFN zinc finger nuclease
  • ZF zinc-finger
  • Each zinc finger module in a ZF array targets three DNA bases.
  • a customized array of individual zinc finger domains is assembled into a ZF protein (ZFP). The resulting ZFP can be linked to a functional domain such as a nuclease.
  • ZF nucleases may be used as alternative programmable nucleases for use in retron-based editing in place of RNA-guide nucleases.
  • ZFN proteins have been extensively described in the art, for example, in Carroll et al.,“Genome Engineering with Zinc-Finger Nucleases,” Genetics, Aug 2011, Vol.188: 773-782; Durai et al.,“Zinc finger nucleases: custom- designed molecular scissors for genome engineering of plant and mammalian cells,” Nucleic Acids Res, 2005, Vol.33: 5978-90; and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering,” Trends Biotechnol.2013, Vol.31: 397-405, each of which are incorporated herein by reference in their entireties.
  • the ZF-linked nuclease is a catalytic domain of the Type IIS restriction enzyme FokI (see Kim et al., PNAS U.S.A.91:883-887, 1994; Kim et al., PNAS U.S.A.93:1156-1160, 1996, both incorporated herein by reference).
  • the ZFN comprises paired ZFN heterodimers, resulting in increased cleavage specificity and/or decreased off-target activity.
  • each ZFN in the heterodimer targets different nucleotide sequences separated by a short spacer (see Doyon et al., Nat.
  • the ZFN comprises a polynucleotide-binding domain (comprising multiple sequence-specific ZF modules) and a polynucleotide cleavage nickase domain.
  • the ZFs are engineered using libraries of two finger modules.
  • strings of two-finger units are used in ZFNs to improve DNA binding specificity from polyzinc finger peptides (see PNAS USA 98: 1437-1441, incorporated herein by reference).
  • the ZFN has more than 3 fingers.
  • the ZFN has 4, 5, or 6 fingers.
  • the ZF modules in the ZFN are separated by one or more linkers to improve specificity.
  • the ZF of the ZFN includes substitutions in the dimer interface of the cleavage domain that prevent homodimerization between ZFs, but allow heterodimers to form.
  • the ZF of the ZFN has a design that retains activity while suppressing homodimerization.
  • the ZFN is any one of the ZF nucleases in Table 1 of Carroll et al., Genetics 188(4):773-782, 2011, incorporated herein by reference.
  • Additional, non-limiting ZFs and AFNz that can be adapted for use in the instant invention include those described in WO2010/065123, WO2000/041566, WO2003/080809, WO2015/143046, WO2016/183298, WO2013/044008, WO2015/031619, WO2017/136049, WO2016/014794, WO2017/091512, WO1995/009233, WO2000/023464, WO2000/042219, WO2002/026960, WO2001/083793; US9428756, US9145565, US8846578, US8524874, US6777185, US6599692, US7235354, US6503717, US7491531, US7943553, US7262054, US8680021, US7705139, US7273923, US6780590, US6785613, US7788044, US7177766, US6453242, US6794136, US7358085, US838376
  • Polynucleotides and vectors capable of expressing one or more of the ZFNs are also provided herein, which can be part of the vector system of the invention.
  • the polynucleotides and vectors can be expressed in a cell, such as a eukaryotic cell, a mammalian cell, or a human cell.
  • a cell such as a eukaryotic cell, a mammalian cell, or a human cell.
  • Suitable vectors, cells and expression systems are described in greater detail elsewhere herein, and can be suitable for use with the TALEs, the meganucleases, and the CRISPR-Cas nucleases.
  • the sequence-specific nuclease is a meganuclease.
  • Meganucleases are a class of sequence-specific endonucleases that recognize large DNA target sites (>12 bp). These proteins can cleave a unique chromosomal sequence without affecting overall genome integrity. Meganucleases create site specific DNA DSBs, and, in the presence of donor DNA, such as one present in the heterologous nucleic acid encompassed by or encoded by the engineered retron of the invention, promotes the integration of the donor DNA at the cleavage site through homologous recombination (HR).
  • HR homologous recombination
  • the meganuclease is a homing endonuclease, which is a widespread class of proteins found in eukaryotes, bacteria and archaea.
  • the meganuclease is of the LAGLIDADG (SEQ ID NO:19953) family of homing endonucleases.
  • the meganuclease is I-Scel, I-Cre-I, I-Dmol, or an engineered or a naturally occurring variant thereof. The hallmark of these proteins is a well conserved LAGLIDADG (SEQ ID NO:19953) peptide motif, termed (do)decapeptide, found in one or two copies.
  • Additional homing nucleases are found at the website of homingendonuclease.net, which provides a database listing basic properties of known LAGLIDADG (SEQ ID NO:19953) homing endonucleases. See also Taylor et al., Nucleic Acids Research 40 (Wl): W110-W116, 2012 (all incorporated herein by reference).
  • specificity (or polynucleotide recognition) of the meganuclease is modified by altering the amino acids within the meganuclease, and/or by fusing other effector domains with the meganuclease.
  • the meganuclease is a megaTAL, which includes a DNA binding domain from a TALE.
  • the meganuclease is engineered to have nickase activity.
  • the sequence-specific nuclease is TnpB, which is a programmable RNA-guided DNA endonuclease. It is believed that TnpB is a functional progenitor of the CRISPR-Cas nucleases.
  • TnpB is a functional progenitor of the CRISPR-Cas nucleases.
  • Transposons are mobile genetic elements that contain only the genes required for their transposition and its regulation. These elements encode the tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition.
  • TnpB has been shown to be a nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif, and TnpB can be reprogrammed to cleave DNA target sites in human cells.
  • TnpB is from D. radiodurans ISDra2 of the IS200/IS605 family.
  • TnpB is from transposon PsiTn554.
  • the sequence-specific nuclease is a TnpB-like protein, such as Fanzor1 or Fanzor2.
  • Fanzor and TnpB proteins share the same conserved amino acid motif in their C-terminal half regions: D-X(125, 275)-[TS]-[TS]-X-X-[C4 zinc finger]-X(5,50)-RD, but are highly variable in their N-terminal regions.
  • Fanzor1 proteins are frequently captured by DNA transposons from different superfamilies including Helitron, Mariner, IS4-like, Sola and MuDr. In contrast, Fanzor2 proteins appear only in some IS607-type elements.
  • the sequence-specific nuclease is IscB.
  • ISC insertion sequences Cas9-like
  • ISC transposon-encoded two nuclease domain-containing proteins are the likely ancestors of the CRISPR-associated Cas9.
  • the homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain.
  • ISC genes are not linked to Cas genes or CRISPR. They represent a distinct group of nonautonomous transposons, with many diverse families of full-length ISC transposons.
  • terminal sequences are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene, and often also encode the TnpB protein containing the RuvC-like endonuclease domain.
  • the terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication.
  • the sequence-specific nuclease is a restriction endonuclease (RE), such as an RE with stringent / long recognition sequence of at least 8 nts.
  • RE restriction endonuclease
  • the RE is a rare-cutter RE with seven and eight base pair recognition sequences.
  • Exemplary rare-cutter RE enzyme include NotI, which cuts after the first GC of a 5 ⁇ -GCGGCCGC-3 ⁇ sequence (SEQ ID NO: 19417).
  • the components of the system – e.g., the retron encoded ncRNA or msDNA in complex with the RT, the sequence-specific nuclease, and the DNA-repair modulating biomolecule, may form multiple complexes in a so-called split complex configuration.
  • the multiple complexes may be brought together to form a functional complex.
  • a first component in the system may be a split protein or domain.
  • a fragment of the split protein or domain may associate with a second component of the system, while another fragment of the split protein or domain may associate with a third component of the system.
  • the split protein or domain is the sequence-specific nuclease, e.g., a CRISPR/Cas effector enzyme (e.g., Cas protein such as Cas9 or Cas12), a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE).
  • a CRISPR/Cas effector enzyme e.g., Cas protein such as Cas9 or Cas12
  • a ZFN e.g., Cas protein such as Cas9 or Cas12
  • TALEN e.g., TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE).
  • the split protein or domain is the reverse transcriptase domain.
  • the split protein or domain is the DNA-repair modulating biomolecule.
  • a first fragment of the sequence-specific nuclease may associate with the reverse transcriptase domain and a second fragment of the sequence-specific nuclease may associate with the DNA-repair modulating biomolecule.
  • the two fragments of the split protein or domain may be brought together (e.g., along with the reverse transcriptase domain and the DNA- repair modulating biomolecule) to form a functional complex.
  • the associations between the parts of the split protein or domain may be through adaptor proteins or linkers described herein (e.g., those used for associating Cas proteins with function domains).
  • the split protein or domain is split in the sense that the two parts of the split protein or domain substantially comprise a functioning split protein or domain. Ideally, the split should always be so that the catalytic domain(s) are unaffected. That split protein or domain may function as a sequence-specific nuclease or it may be a dead-Cas which is essentially an RNA-binding protein with very little or no catalytic activity, due to typically mutation(s) in its catalytic domains. [00648] Each fragment of the split protein or domain may be fused to a dimerization partner.
  • rapamycin sensitive dimerization domains enables a chemically inducible split protein or domain for temporal control of the split protein or domain’s activity.
  • the split protein or domain can thus be rendered chemically inducible by being split into two fragments and that rapamycin-sensitive dimerization domains may be used for controlled reassembly of the split protein or domain.
  • the two parts of the split protein or domain can be thought of as the N’ terminal part and the C’ terminal part of the split protein or domain.
  • the fusion is typically at the split point of the split protein or domain.
  • the C’ terminal of the N’ terminal part of the split protein or domain is fused to one of the dimer halves, whilst the N’ terminal of the C’ terminal part is fused to the other dimer half.
  • the split protein or domain does not have to be split in the sense that the break is newly created.
  • the split point is typically designed in silico and cloned into the constructs.
  • the two parts of the split protein or domain, the N’ terminal and C’ terminal parts form a full split protein or domain, comprising preferably at least 70% or more of the wildtype amino acids (or nucleotides encoding them), at least 80% or more, at least 90% or more, at least 95% or more, and at least 99% or more of the wildtype amino acids (or nucleotides encoding them).
  • the dimer may be a homodimer or a heterodimer.
  • the protein components of the system – e.g., the RT, the sequence-specific nuclease (a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)), and the DNA-repair modulating biomolecule may further comprise one or more additional functional domains.
  • the functional domain comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • one or more C-terminal or N-terminal NLSs are attached.
  • a C-terminal NLS is attached for expression and nuclear targeting in eukaryotic cells, e.g., human cells.
  • the NLS(s) may be at a location that is not at the C-terminus or N-terminus, for example, the NLS(s) may be between two polypeptides.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen; the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS); the c-myc NLS; the hRNPAl M9 NLS; the NLS of the IBB domain from importin-alpha; the NLS of the myoma T protein; the NLS of human p53; the NLS of mouse c- abl IV; the NLS of the influenza virus NS1; the NLS of the Hepatitis virus delta antigen; the NLS of the mouse Mxl protein; the NLS of the human poly(ADP-ribose) polymerase; and the NLS of the steroid hormone receptors (human) glucocorticoid.
  • nucleoplasmin e.g., the nucleoplasmin bipartite NLS
  • the c-myc NLS e.g., the nucleoplasm
  • the functional domain comprises at least two NLS domains.
  • the one or more NLS domain(s) may be positioned at or near or in proximity to a terminus of a polypeptide and, if two or more NLSs, each of the two may be positioned at or near or in proximity to a terminus of the polypeptide.
  • the fusion between the two domains (such as the RT and Cas enzyme, or the Cas and the DNA-repair modulating biomolecule) may be linked through a linker.
  • a “linker” as used herein includes a peptide which joins two proteins or domains to form a fusion protein. Generally, such molecules have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins / domains. However, in certain embodiments, the linker may be selected to influence some property of the linker and/or the fusion protein such as the folding, net charge, or hydrophobicity of the linker. Suitable linkers for use in the present disclosure are well-known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linker may also be a covalent bond (carbon-carbon bond or carbon-heteroatom bond).
  • the linker is used to separate the sequence-specific nuclease (a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)) and the RT and/or the DNA-repair modulating biomolecule by a distance sufficient to ensure that each protein domain retains its required functional property.
  • sequence-specific nuclease a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)
  • RE restriction endonuclease
  • Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure.
  • the linker can be a chemical moiety which can be monomeric, dimeric, multimeric or polymeric.
  • the linker comprises amino acids. Typical amino acids in flexible linkers include Gly, Asn and Ser. Accordingly, in particular embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near neutral amino acids, such as Thr and Ala, also may be used in the linker sequence.
  • the linker comprises a G 4 S linker with 3, 6, 9, or 12 repeats.
  • the linker is one disclosed in Maratea et al., Gene 40: 39- 46, 1985; Murphy et al., PNAS USA 83: 8258-62, 1986; US4,935,233; or US4,751,180, all incorporated by reference.
  • the linker comprises a GlySer linker such as GGS, GGGS (SEQ ID NO:19947), GSG, GGGGS (SEQ ID NO:19948), optionally with repeats of 3 (such as (GGS) 3 , (SEQ ID NO:19949) (GGGGS) 3 ) (SEQ ID NO:19950), 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more, to provide suitable lengths.
  • the linker comprises (GGGGS) 3-15 (SEQ ID NO:19951), such as (GGGGS) 3-11 (SEQ ID NO:19951), e.g., GGGGS (SEQ ID NO:19951) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 repeats.
  • the linker comprises LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19418).
  • the linker is an XTEN linker.
  • N-and/or C-terminal NLSs also function as linker (e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NO: 19420)).
  • the gRNA and the various nucleases and fusions thereof can be provided in the form of a protein, optionally where the nuclease is complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector).
  • the RNA-guided nuclease and the gRNA are both provided by vectors.
  • RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences.
  • Codon usage may be optimized to improve production of the engineered retron e.g., retron reverse transcriptase, ncRNA and/or RNA-guided nuclease in a particular cell or organism.
  • a nucleic acid encoding an ncRNA, RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a the particular cell such as a eukaryotic cell (e.g., yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell), or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • a nucleic acid encoding the reverse transcriptase or ncRNA is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.
  • the recombinant retron-based editing system described herein contemplates fusion proteins comprising a programmable nuclease (PN) and a RT, optionally joined by a linker.
  • PN programmable nuclease
  • RT e.g., retron RTs of Table A
  • the RT is joined to the N-terminus of the PN.
  • the RT is joined to the C-terminus of the PN.
  • the fusion proteins may comprise any suitable structural configuration.
  • the fusion protein may comprise from the N-terminus to the C- terminus direction, a PN fused to a RT.
  • the fusion protein may comprise from the N-terminus to the C-terminus direction, a RT fused to a NP.
  • the fused domain may optionally be joined by a linker, e.g., an amino acid sequence.
  • Retron-based gene editing systems [00670] The present disclosure relates to novel genome editing systems that include retrons, including retron RT and ncRNAs.
  • the editing systems comprise: (a) one or more retron RT polypeptide sequences comprising at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% sequence identity to any one of the amino acid sequences of Table A, or a polynucleotide sequence encoding same (as also provided in Table A); (b) (a) one or more retron ncRNA polynucleotide sequences comprising at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% sequence identity to any one of the amino acid sequences of Table B, wherein in one embodiment the retron RT and ncRNA are cognate pairs; (a) one or more
  • the retron-based gene editing systems may comprise one or more additional accessory proteins having genome modifying functions, including recombinases, invertases, nucleases, polymerases, ligases, deaminases, reverse transcriptases, or epigenetic modifying functions.
  • the accessory proteins may be provided separately.
  • the accessory proteins may be fused to a retron component (e.g., fused to a retron RT), optionally with a linker.
  • the genome editing system may comprise a guide RNA, which hybridizes to one or more targeted polynucleotide sequence.
  • the guide RNA of the genome editing system comprises 12-40 nucleotides.
  • the targeted polynucleotide sequence comprises one or more relaxed PAM recognition domains. Jacobsen, Thomas et al.
  • the editing system to recognize altered PAM recognition domains for genome editing.
  • the programmable polypeptide component recognizes one or more non-canonical PAM sequence in the targeted polynucleotide sequence, the PAM upstream of the crRNA-complementary DNA sequence on the non-target strand.
  • the gRNA has a seed sequence of eight nucleotides, located at the 5′ end of the spacer, and is proximal to the PAM sequence on the targeted polynucleotide sequence.
  • the one or more polypeptide sequences and the one or more polynucleotide sequences comprising a cognate guide RNA of the genome editing system form a ribonucleoprotein complex.
  • the programmable nuclease is a Type V enzyme (e.g., Cas12a)
  • the one or more polypeptide sequences of the genome editing system comprise: • one or more ⁇ -helical recognition lobe (REC) and a nuclease lobe (NUC); • a Wedge (WED), ⁇ -helical recognition lobe (REC), PAM-interacting (PI), • RuvC nuclease, Bridge Helix (BH) and NUC domains; or • one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains.
  • REC ⁇ -helical recognition lobe
  • NUC nuclease lobe
  • WED Wedge
  • REC ⁇ -helical recognition lobe
  • PI
  • the one or more polypeptide sequences of the genome editing system comprise: • An alpha-helical lobe and • A nuclease lobe that comprises two nuclease domains (the RuvC domain which cleaves the non-target DNA strand and the HNH domain that cleaves the target strand).
  • the molecular weight of the programmable nuclease component is characterized in its molecular weight to be about 50 kDa – 100kDa, 100 kDa – 200kDa, 200kDa – 500kDa.
  • the polypeptide sequences comprise at least one activity selected from endonuclease activity; endoribonuclease activity, or RNA-guided DNase activity.
  • the cognate guide RNA and the Cas12a protein modifies the targeted polynucleotide sequence of a host cell genome.
  • the targeted polynucleotide sequence is modified by an insertion, deletion or alteration of one or more base pairs at the targeted polynucleotide sequence in the host cell genome.
  • the genome editing system is characterized in enhanced efficiency and precision of site-directed integration.
  • the efficiency and precision of site-directed integration enabled by genome editing system is enhanced by staggered overhangs on the donor nucleic acid sequence.
  • the targeted polynucleotide sequence is double-stranded and contains a 5′ overhang wherein the overhang preferably comprises five nucleotides.
  • the polypeptide of the genome editing system comprises one or more mutations. More preferably, the mutation encodes a nuclease-deficient polypeptide. In various embodiments, the genome editing system comprises a fusion of one or more deaminases to the nuclease deficient polypeptide.
  • the one or more deaminases of the genome editing system is selected from adenine deaminase or cytosine deaminase.
  • adenine deaminase or cytosine deaminase Use of cytidine deaminase and adenosine deaminase base editing is disclosed in U.S. Pat. No.9,840,699.
  • One approach is to produce an Cas12a fusion protein, preferably an inactive or nickase variant) and a base- editing enzyme or the active domain of a base editing enzyme. Cytidine deaminase and adenosine deaminase base editing is disclosed in U.S. Pat. No.9,840,699.
  • the compositions comprise contacting a targeted polynucleotide sequence with a fusion protein comprising an Cas12a and one or more base-editing polypeptide such as a deaminase; and a gRNA targeting the fusion protein to the targeted polynucleotide sequence of the DNA strand.
  • a fusion protein comprising an Cas12a and one or more base-editing polypeptide such as a deaminase
  • a gRNA targeting the fusion protein to the targeted polynucleotide sequence of the DNA strand.
  • the fusion of one or more deaminases to the nuclease deficient polypeptide of the Cas12a genome editing system enables base editing on DNA and/or RNA.
  • the system modifies one or more nucleobase on DNA and RNA.
  • the system enables multiplexed gene editing.
  • the genome editing system comprises a single crRNA.
  • the system enables targeting multiple genes simultaneously.
  • the programmable polypeptide is operably linked to a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the programmable polypeptide comprises an NLS on the N-terminus or the C-terminus or both or multiple NLS on the Cas12a polypeptide.
  • the polypeptide linked to the NLS further comprises crRNA to form a ribonucleoprotein complex.
  • polypeptide comprises one or more NLS repeats at either N- or C- terminus of the polypeptide.
  • the one or more polypeptide sequences of the genome editing system comprises a modification, wherein the modification comprises a nuclease- deficient polypeptide (dCas).
  • the guide RNA of the genome editing system comprises a prime editing guide RNA (pegRNA).
  • the pegRNA of the genome editing system hybridizes to a targeted polynucleotide sequence and acts as a primer to the one or more reverse transcriptases. More preferably, the pegRNA of the genome editing system binds to the nicked strand for initiation of repair through a reverse transcriptase using the repair template.
  • the nuclease-deficient polypeptide of the genome editing system comprises a nickase activity.
  • the genome editing system comprises fusion of one or more reverse transcriptases to the nuclease deficient Cas (dCas).
  • the fusion of one or more reverse transcriptases is selected from Optional components/modifications Donor templates [00685]
  • the compositions and systems herein may further comprise one or more donor templates for use in editing.
  • the donor template may comprise one or more polynucleotides.
  • the donor template may comprise coding sequences for one or more polynucleotides.
  • the donor template may be a DNA template.
  • the donor template may become integrated into the genome after a targeted cut by a programmable nuclease cutter described herein through cellular repair machinery including HDR and NHEJ.
  • the HDR donor is formed from reverse transcription of the ncRNA and forms the RT-DNA portion thereof.
  • the donor template which may be integrated into a ncRNA and expressed as an RT-DNA product, may be used for editing the target polynucleotide.
  • the donor polynucleotide comprises one or more mutations to be introduced into the target polynucleotide.
  • mutations include substitutions, deletions, insertions, or a combination thereof.
  • the mutations may cause a shift in an open reading frame on the target polynucleotide.
  • the donor template alters a stop codon in the target polynucleotide.
  • the donor template may correct a premature stop codon. The correction may be achieved by deleting the stop codon or introduces one or more mutations to the stop codon.
  • the donor template addresses loss of function mutations, deletions, or translocations that may occur, for example, in certain disease contexts by inserting or restoring a functional copy of a gene, or functional fragment thereof, or a functional regulatory sequence or functional fragment of a regulatory sequence.
  • a functional fragment refers to less than the entire copy of a gene by providing sufficient nucleotide sequence to restore the functionality of a wild type gene or non-coding regulatory sequence (e.g. sequences encoding long non-coding RNA).
  • the systems disclosed herein may be used to replace a single allele of a defective gene or defective fragment thereof.
  • the systems disclosed herein may be used to replace both alleles of a defective gene or defective gene fragment.
  • a “defective gene” or “defective gene fragment” is a gene or portion of a gene that when expressed fails to generate a functioning protein or non-coding RNA with functionality of a corresponding wild-type gene.
  • these defective genes may be associated with one or more disease phenotypes.
  • the defective gene or gene fragment is not replaced but the systems described herein are used to insert donor templates that encode gene or gene fragments that compensate for or override defective gene expression such that cell phenotypes associated with defective gene expression are eliminated or changed to a different or desired cellular phenotype.
  • the donor template may include, but not be limited to, genes or gene fragments, encoding proteins or RNA transcripts to be expressed, regulatory elements, repair templates, and the like.
  • the donor templates may comprise left end and right end sequence elements that function with transposition components that mediate insertion.
  • the donor template manipulates a splicing site on the target polynucleotide.
  • the donor template disrupts a splicing site. The disruption may be achieved by inserting the polynucleotide to a splicing site and/or introducing one or more mutations to the splicing site.
  • the donor template may restore a splicing site.
  • the polynucleotide may comprise a splicing site sequence.
  • the donor template to be inserted may has a size from 10 base pair or nucleotides to 50 kb in length, e.g., from 50 to 40k, from 100 and 30 k, from 100 to 10000, from 100 to 300, from 200 to 400, from 300 to 500, from 400 to 600, from 500 to 700, from 600 to 800, from 700 to 900, from 800 to 1000, from 900 to from 1100, from 1000 to 1200, from 1100 to 1300, from 1200 to 1400, from 1300 to 1500, from 1400 to 1600, from 1500 to 1700, from 600 to 1800, from 1700 to 1900, from 1800 to 2000 base pairs (bp) or nucleotides in length.
  • the heterologous nucleic acid sequence is a donor DNA template that can be integrated into a host genome via HDR. In other embodiments, the heterologous nucleic acid sequence is a donor DNA template that can be integrated into a host genome via NHEJ. [00691] In certain embodiments, the heterologous nucleic acid comprises or encodes a donor / template sequence, wherein the donor / template corrects / repairs / removes a mutation at the target genome site. For example, the mutation may be a mutated exon in a disease gene.
  • the donor / template may encode or comprises a functional DNA element, such as a promoter, an enhancer, a protein binding sequence, a methylation site, or a homology region for assisting gene editing, etc.
  • a functional DNA element such as a promoter, an enhancer, a protein binding sequence, a methylation site, or a homology region for assisting gene editing, etc.
  • donor DNA or “donor DNA template” it is meant a DNA segment (can be single stranded or double stranded DNA) to be inserted at a site cleaved by a gene-editing nuclease (e.g., a Cas9 or Cas12a nuclease) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like).
  • a gene-editing nuclease e.g., a Cas9 or Cas12a nuclease
  • the donor DNA template can contain sufficient homology to a genomic sequence at the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. In the case of repair by NHEJ, no homology is needed on the donor DNA template against the site to which it targets editing.
  • the donor template can be integrated into a ncRNA of a retron and expressed as a RT product as an RT-DNA.
  • Donor DNA template can be of any length, e.g., 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
  • a suitable donor DNA template can be from 50 nucleotides to 100 nucleotides, from 100 nucleotides to 500 nucleotides, from 500 nucleotides to 1000 nucleotides, from 1000 nucleotides to 5000 nucleotides, or from 5000 nucleotides to 10,000 nucleotides, or more than 10,000 nucleotides, in length.
  • the donor DNA template comprises a first homology arm and a second homology arm.
  • the first homology arm is at or near the 5’ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a first nucleotide sequence in a target nucleic acid.
  • the second homology arm is at or near the 3’ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a second nucleotide sequence in the target nucleic acid.
  • the first and second homology arms can each independently have a length of from about 10 nucleotides to 400 nucleotides; e.g., from 10 nucleotides (nt) to 15 nt, from 15 nt to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, from 45 nt to 50 nt, from 50 nt to 75 nt, from 75 nt to 100 nt, from 100 nt to 125 nt, from 125 nt to 150 nt, from 150 nt to 175 nt, from 175 nt to 200 nt
  • the donor DNA template is used for editing the target nucleotide sequence.
  • the donor DNA template comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or a combination thereof.
  • the mutation causes a shift in an open reading frame on the target polynucleotide.
  • the donor polynucleotide alters a stop codon in the target polynucleotide. In certain embodiments, the donor polynucleotide corrects a premature stop codon.
  • the correction can be achieved by deleting the stop codon, or by introducing one or more sequence changes to alter the stop codon to a codon.
  • the donor polynucleotide addresses loss of function mutations, deletions, or translocations that may occur, for example, in certain disease contexts by inserting or restoring a functional copy of a gene, or functional fragment thereof, or a functional regulatory sequence or functional fragment of a regulatory sequence.
  • a functional fragment includes a fragment less than the entire copy of a gene but otherwise provides sufficient nucleotide sequence to restore the functionality of a wild type gene or non-coding regulatory sequence (e.g., sequences encoding long non-coding RNA).
  • the donor DNA template may be used to replace a single allele of a defective gene or defective fragment thereof. In another embodiment, the donor DNA template is used to replace both alleles of a defective gene or defective gene fragment.
  • a “defective gene” or “defective gene fragment” is a gene or portion of a gene that when expressed, fails to generate a functioning protein or non-coding RNA with functionality of the corresponding wild-type gene. [00698] In certain example embodiments, these defective genes may be associated with one or more disease phenotypes.
  • the defective gene or gene fragment is not replaced but the heterologous nucleic acid is used to insert donor polynucleotides that encode gene or gene fragments that compensate for or override defective gene expression such that cell phenotypes associated with defective gene expression are eliminated or changed to a different or desired cellular phenotype.
  • This can be achieved by including the coding sequence of a therapeutic protein, such as a therapeutic antibody or functional fragment thereof, or a wild- type version of a defective protein associated with one or more disease phenotypes.
  • the donor may include, but not be limited to, genes or gene fragments, encoding proteins or RNA transcripts to be expressed, regulatory elements, repair templates, and the like.
  • the donor polynucleotides may comprise left end and right end sequence elements that function with transposition components that mediate insertion.
  • the donor DNA template manipulates a splicing site on the target polynucleotide.
  • the donor DNA template disrupts a splicing site. The disruption may be achieved by inserting the polynucleotide to a splicing site and/or introducing one or more mutations to the splicing site.
  • the donor polynucleotide may restore a splicing site.
  • the polynucleotide may comprise a splicing site sequence.
  • the donor DNA template to be inserted has a size from 10 bp to 50 kb in length, e.g., from 50 bp to ⁇ 40kb, from 100 bp to ⁇ 30 kb, from 100 bp to ⁇ 10 kb, from 100 bp to 300 bp, from 200 bp to 400 bp, from 300 bp to 500 bp, from 400 bp to 600 bp, from 500 bp to 700 bp, from 600 bp to 800 bp, from 700 bp to 900 bp, from 800 bp to 1000 bp, from 900 bp to 1100 bp, from 1000 bp to 1200 bp, from 1100 bp to 1300 bp, from 1200 bp to 1400 bp, from 1300 bp to 1500 bp, from 1400 bp to 1600 bp, from 1500 bp to 1700 bp,
  • the homologous arm on one or both ends of the sequence to be inserted is independently about 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, or 150 bp.
  • the first homology arm and the second homology arm of the donor DNA flank a nucleotide sequence (“a nucleotide sequence of interest” or “an intervening nucleotide sequence”) that is to be introduced into a target nucleic acid.
  • the nucleotide sequence of interest can comprise: i) a nucleotide sequence encoding a polypeptide of interest; ii) a nucleotide sequence encoding an exon of a gene; iii) a promoter sequence; iv) an enhancer sequence; v) a nucleotide sequence encoding a non-coding RNA; or vi) any combination of the foregoing.
  • the donor DNA can provide for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.
  • the donor DNA can be used to add, e.g., insert or replace, nucleic acid material to a target DNA (e.g.
  • a tag e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.
  • a regulatory sequence e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, enhancer, etc.
  • a nucleic acid sequence e.g., introduce a mutation
  • the donor DNA can be used to modify DNA in a site-specific, i.e. “targeted”, way; for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapy, e.g. to treat a disease; or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of pluripotent stem cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.
  • the donor DNA comprises a nucleotide sequence encoding a polypeptide of interest.
  • Polypeptides of interest include, e.g., a) functional versions of a polypeptide that comprises one or more amino acid substitutions, insertions, and/or deletions and that exhibits reduced function, e.g., where the reduced function is associated with or causes a pathological condition; b) fluorescent polypeptides; c) hormones; d) receptors for ligands; e) ion channels; f) neurotransmitters; g) and the like.
  • the donor DNA comprises a nucleotide sequence that encodes a wild-type protein that is lacking in the recipient cell.
  • the donor DNA encodes a wild type factor (e.g.
  • the donor DNA comprises a nucleotide sequence that encodes a therapeutic antibody.
  • the donor DNA comprises a nucleotide sequence that encodes an engineered protein or receptor.
  • the engineered receptor is a T cell receptor (TCR), a natural killer (NK) receptor (NKR), or a B cell receptor (BCR).
  • TCR T cell receptor
  • NK natural killer
  • BCR B cell receptor
  • the engineered TCR or NKR targets a cancer marker (e.g., a polypeptide that is expressed (e.g., over-expressed) on the surface of a cancer cell).
  • the donor DNA comprises a nucleotide sequence that encodes a chimeric antigen receptor (CAR).
  • CAR targets a cancer marker.
  • Donor DNAs encoding CAR, TCR, and/or NCR proteins may be folded into DNA origami structures (DNA nanostructures) and delivered into T cells or NK cells in vitro or in vivo.
  • Non-limiting examples of polypeptides that can be encoded by a donor DNA include, e.g., IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11), INS (insulin), CRP (C -reactive protein, pentraxin-related), PDGFRB (platelet- derived growth factor receptor, beta polypeptide), CCNA2 (cyclin A2), PDGFB
  • ACE angiotensin I converting enzyme peptidyl-dipeptidase A 1)
  • TNF tumor necrosis factor
  • IL6 interleukin 6 (interferon, beta 2)
  • STN statin
  • SERPINE1 serotonin peptidase inhibitor
  • clade E nonin, plasminogen activator inhibitor type 1
  • ALB albumin
  • ADIPOQ adiponectin, C1Q and collagen domain containing
  • APOB apolipoprotein B (including Ag(x) antigen)
  • APOE apolipoprotein E
  • LEP laeptin
  • MTHFR 5,10- methylenetetrahydrofolate reductase (NADPH)
  • APOA1 apolipoprotein A-I
  • EDN1 endothelin 1
  • NPPB natriuretic peptide precursor B
  • NOS3 nitric oxide synthase 3
  • GNRH1 gonadotropin-releasing hormone 1 (luteinizing- releasing hormone)
  • PAPPA pregnancy-associated plasma protein A, pappalysin 1
  • ARR3 arrestin 3, retinal (X-arrestin)
  • NPPC natriuretic peptide precursor C
  • AHSP alpha hemoglobin stabilizing protein
  • PTK2 PTK2 protein tyrosine kinase 2
  • IL13 interleukin 13
  • MTOR mechanistic target of rapamycin (serine/threonine kinase)
  • ITGB2 integratedin, beta 2 (complement component 3 receptor 3 and 4 subunit)
  • GSTT1 glutthione S-transfcrase theta 1
  • IL6ST interleukin 6 signal transducer (gpl30, oncostatin M receptor)
  • CPB2 carboxypeptidase B2 (plasma)
  • CYP1A2 cytochrome P
  • CAMP cathelicidin antimicrobial peptide
  • ZC3H12A zinc finger CCCH-type containing 12A
  • AKR1B1 aldo-keto reductase family 1, member B1 (aldose reductase)
  • DES desmin
  • MMP7 matrix metallopeptidase 7 (matrilysin, uterine)
  • AHR aryl hydrocarbon receptor
  • CSF1 colony stimulating factor 1 (macrophage)
  • HDAC9 histone deacetylase 9
  • CTGF connective tissue growth factor
  • KCNMA1 potassium large conductance calcium- activated channel, subfamily M, alpha member 1
  • UGT1A UDP glucuronosyltransferase 1 family, polypeptide A complex locus
  • PRKCA protein kinase C, alpha
  • COMT catechol-b- methyltransf erase
  • S100B S100 calcium binding protein B
  • the donor DNA encodes a wild-type version of any of the foregoing polypeptides; i.e., the donor DNA can encode a “normal” version that does not include a mutation(s) that results in reduced function, lack of function, or pathogenesis.
  • the donor DNA comprises a nucleotide sequence encoding a fluorescent polypeptide.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t- HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kae
  • fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909), and the like.
  • the donor DNA encodes an RNA, e.g., an siRNA, a microRNA, a short hairpin RNA (shRNA), an anti-sense RNA, a riboswitch, a ribozyme, an aptamer, a ribosomal RNA, a transfer RNA, and the like.
  • a donor DNA can include, in addition to a nucleotide sequence encoding one or more gene products (e.g., an RNA and/or a polypeptide), one or more transcriptional control elements, e.g., a promoter, an enhancer, and the like. In some cases, the transcriptional control element is inducible.
  • the promoter is reversible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in a eukaryotic cell. In some cases, the promoter is a cell type- specific promoter. In some cases, the promoter is a tissue-specific promoter. [00711]
  • the nucleotide sequence of the donor DNA is typically not identical to the target nucleic acid (e.g., genomic sequence) that it replaces.
  • the donor DNA may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the target nucleic acid (e.g., genomic sequence), so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease- causing base pair or a non-disease-causing base pair).
  • the donor DNA comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.
  • Donor DNA may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest (the target nucleic acid) and that are not intended for insertion into the DNA region of interest (the target nucleic acid).
  • the homologous region(s) of a donor sequence will have at least 50% sequence identity to a target nucleic acid (e.g., a genomic sequence) with which recombination is desired. In certain cases, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
  • the donor DNA may comprise certain nucleotide sequence differences as compared to the target nucleic acid (e.g., genomic sequence), where such difference include, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor DNA at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus).
  • nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein).
  • the donor DNA will include one or more nucleotide sequences to aid in localization of the donor to the nucleus of the recipient cell or to aid in the integration of the donor DNA into the target nucleic acid.
  • the donor DNA may comprise one or more nucleotide sequences encoding one or more nuclear localization signals and the like (Frietas et al (2009) Cun- Genomics 10:550-7).
  • the donor DNA will include nucleotide sequences to recruit DNA repair enzymes to increase insertion efficiency.
  • Fiuman enzymes involved in homology directed repair include MRN-CtIP, BLM-DNA2, Exol, ERCC1, Rad51, Rad52, Ligase 1, RoIQ, PARP1, Ligase 3, BRCA2, RecQ/BLM-ToroIIIa, RTEL, Ro ⁇ d, and Ro ⁇ h (Verma and Greenburg (2016) Genes Dev.30 (10): 1138-1154).
  • the donor DNA is delivered as reconstituted chromatin (Cruz- Becerra and Kadonaga (2020) eLife 2020;9:e55780 DOI: 10.7554/eLife.55780).
  • the ends of the donor DNA are protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art.
  • one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or self complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.
  • Linkers [00714]
  • the polypeptides of the retron-based gene editing system are coupled to to one another other to one or more accessory functions by a linker.
  • Such accessory functions can include deaminases, nucleases, reverse transcriptases, and recombinases.
  • a retron RT may be linked to a programmable nuclease, such as, a Type II or Type V CRISPR nuclease.
  • the term linker as used in reference to a fusion protein refers to a molecule which joins the proteins to form a fusion protein. Generally, such molecules have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins. However, in one embodiment, the linker may be selected to influence some property of the linker and/or the fusion protein such as the folding, net charge, or hydrophobicity of the linker.
  • Suitable linkers for use in the methods of the present invention are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure.
  • the linker can be a chemical moiety which can be monomeric, dimeric, multimeric or polymeric.
  • the linker comprises amino acids. Typical amino acids in flexible linkers include Gly, Asn and Ser.
  • the linker comprises a combination of one or more of Gly, Asn and Ser amino acids.
  • GlySer linkers may be based on repeating units of GGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeating units, including but not limited to:
  • GlySer linkers may be based on repeating units of GSG, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeating units, including but not limited to:
  • GlySer linkers may be based on repeating units of GGGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeating units, including but not limited to:
  • GlySer linkers may be based on repeating units of GGGGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeating units, including but not limited to:
  • LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19526) is used as a linker.
  • the linker is an XTEN linker, which is TCGGGATCTGAGACGCCTGGGACCTCGGAATCGGCTACGCCCGAAAGT (SEQ ID NO: 19527).
  • Cas12a polypeptide is linked C-terminally to the N- terminus of a deaminase protein or its catalytic domain by means of an LEPGEKPYKCPECGKSFSQSGALTRHQRTHTRLEPGEKPYKCPECGKSFSQSGALTRHQR THTRLEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19528) linker.
  • N-and C-terminal NLSs can also function as linker (e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NO: 19420)).
  • linkers is intended to be non-limiting and includes any combinations of the above linkers or heterologous combinations of repeating GlySer linkers.
  • the linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length.
  • the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like.
  • the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).
  • the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker.
  • the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.).
  • the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid.
  • the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.).
  • the linker comprises a monomer, dimer, or polymer of aminoHEXAnoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cycloHEXAne). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring.
  • Ahx aminoHEXAnoic acid
  • the linker may included functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker.
  • a nucleophile e.g., thiol, amino
  • Any electrophile may be used as part of the linker.
  • Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
  • the linker can be, for example, a cleavable linker or protease-sensitive linker.
  • the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof.
  • This family of self-cleaving peptide linkers referred to as 2A peptides, has been described in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE 6:e18556).
  • the linker is an F2A linker.
  • the linker is a GGGS linker.
  • the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker- domain.
  • Cleavable linkers known in the art may be used in connection with the disclosure.
  • Exemplary such linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750).
  • linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750).
  • linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750).
  • linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750).
  • other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure).
  • polycistronic constructs mRNA
  • nuclear localization domains [00726]
  • the gene editing systems or any of the components thereof may fused to one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • NLSs nuclear localization sequences
  • a gene editor component e.g., a nucleic acid programmable DNA binding protein or an editing accessory protein
  • each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • an editor component polypeptide comprises at most 6 NLSs.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 19399); the NLS from nucleoplasmin (e.g.
  • the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 19529); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 19530) or RQRRNELKRSP (SEQ ID NO: 19531); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 19532); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 19533) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 19400) and PPKKARED (SEQ ID NO: 19534) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 19535) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 19536) of mouse c-abl IV
  • the one or more NLSs are of sufficient strength to drive accumulation of a polypeptide of interest (e.g., retron RT) in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the polypeptide of interest (e.g., retron RT), the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to a polypeptide of interest (e.g., retron RT), such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI).
  • a means for detecting the location of the nucleus e.g., a stain specific for the nucleus such as DAPI.
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay.
  • Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of complex formation (e.g., assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by complex formation and/or polypeptide activity), as compared to a control no exposed to the polypeptide or complex, or exposed to a polypeptide lacking the one or more NLSs.
  • an assay for the effect of complex formation e.g., assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by complex formation and/or polypeptide activity
  • the codon optimized polypeptide proteins comprise an NLS attached to the C-terminal of the protein.
  • other localization tags may be fused to the polypeptide, such as without limitation for localizing the polypeptide to particular sites in a cell, such as organelles, such as mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear or cellular) membranes, ribosomes, nucleolus, ER, cytoskeleton, vacuoles, centrosome, nucleosome, granules, centrioles, etc.
  • organelles such as mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear or cellular) membranes, ribosomes, nucleolus, ER, cytoskeleton, vacuoles, centrosome, nucleosome, granules, centrioles, etc.
  • NLS nuclear localization signal
  • At least one or more C-terminal or N-terminal NLSs are attached (and hence nucleic acid molecule(s) coding for the polypeptide can include coding for NLS(s) so that the expressed product has the NLS(s) attached or connected).
  • a C- terminal NLS is attached for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells.
  • the invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest.
  • the nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers.
  • the one or more aptamers may be capable of binding a bacteriophage coat protein.
  • the fusion proteins comprising a polypeptide of interest (e.g., retron RT) and another accessory protein (e.g., nuclease) contains one or more nuclear localization signals is selected or derived from SV40, c-Myc or NLP-1.
  • the NLS examples above are non-limiting.
  • proteins and/or fusions contemplated herein may comprise any known NLS sequence, including any of those described in Cokol et al.,“Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415 and Freitas et al.,“Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference. [00732] In various embodiments, any of the polypeptide components of the retron-based editing systems may be engineered with one or more nuclear localization signals, which help promote translocation of a protein into the cell nucleus.
  • the polypeptides of the retron-based editing system may comprise any known NLS sequence, including any of those described in Cokol et al.,“Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415 and Freitas et al.,“Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference. [00733]
  • the polypeptides disclosed herein may include one or more, preferably, at least two nuclear localization signals.
  • the NLSs may be any known NLS sequence in the art.
  • the NLSs may also be any future-discovered NLSs for nuclear localization.
  • the NLSs also may be any naturally-occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations).
  • the term“nuclear localization sequence” or“NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., International PCT application PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference.
  • an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 19399).
  • Another representative nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell in which the sequence is expressed.
  • a nuclear localization signal is predominantly basic, can be positioned almost anywhere in a protein's amino acid sequence, generally comprises a short sequence of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem.273: 14731-37, incorporated herein by reference) to eight amino acids, and is typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274: 11- 16, incorporated herein by reference).
  • Nuclear localization signals often comprise proline residues.
  • a variety of nuclear localization signals have been identified and have been used to effect transport of biological molecules from the cytoplasm to the nucleus of a cell. See, e.g., Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A.89:7442-46; Moede et al., (1999) FEBS Lett.461:229-34, which is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins.
  • NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 19399); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXKKKL) (SEQ ID NO: 19419); and (iii) noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991).
  • NLS nuclear localization signals appear at various points in the amino acid sequences of proteins. NLS’s have been identified at the N-terminus, the C-terminus and in the central region of proteins. Thus, the disclosure provides polypeptides that may be modified with one or more NLSs at the C-terminus, the N-terminus, as well as at in internal region of a polypeptide (including a fusion protein). [00736] The present disclosure contemplates any suitable means by which to modify a polypeptide to include one or more NLSs.
  • a polypeptide e.g., a programmable nuclease
  • a polypeptide-NLS fusion construct may be engineered to express with a translationally fused NLS at its N-terminus or its C-terminus (or both), i.e., to form a polypeptide-NLS fusion construct.
  • the NLSs may include various amino acid linkers or spacer regions encoded between a polypeptide and the N-terminally, C-terminally, or internally-attached NLS amino acid sequence, e.g, and in the central region of proteins.
  • the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a polypeptide and one or more NLSs.
  • the retron-based editing system or a component thereof may comprise a polypeptide tag, such as an affinity tag (chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), SBP-tag, Strep-tag, AviTag, Calmodulin-tag); solubilization tag; chromatography tag (polyanionic amino acid tag, such as FLAG-tag); epitope tag (short peptide sequences that bind to high-affinity antibodies, such as V5-tag, Myc-tag, VSV-tag, Xpress tag, E-tag, S-tag, and HA-tag); fluorescence tag (e.g., GFP).
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • SBP-tag Strep-tag
  • AviTag AviTag
  • Calmodulin-tag Calmodulin-tag
  • solubilization tag solubilization
  • the retron-based editing system peptide may comprise an amino acid tag, such as one or more lysines, histidines, or glutamates, which can be added to the polypeptide sequences (e.g., at the N-terminal or C-terminal ends). Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • Protein and amino acid tags are peptide sequences genetically grafted onto a recombinant protein. Sequence tags are attached to proteins for various purposes, such as peptide purification, identification, or localization, for use in various applications including, for example, affinity purification, protein array, western blotting, immunofluorescence, and immunoprecipitation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal residues
  • the nucleic acid components (e.g., guide RNAs or retron ncRNAs) of the retron-based editing systems may further comprise a functional structure designed to improve nucleic acid component molecule structure, architecture, stability, genetic expression, or any combination thereof.
  • a structure can include an aptamer.
  • Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990, 249:505-510).
  • Nucleic acid aptamers can for example be selected from pools of random- sequence oligonucleotides, with high binding affinities and specificities for a wide range of biomedically relevant targets, suggesting a wide range of therapeutic utilities for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington.
  • aptamers as therapeutics. Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a wide range of uses for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. "Nanotechnology and aptamers: applications in drug delivery.” Trends in biotechnology 26.8 (2008): 442-449; and, Hicke BJ, Stephens AW. “Escort aptamers: a delivery service for diagnosis and therapy.” J Clin Invest 2000, 106:923-928.).
  • RNA aptamers may also be constructed that function as molecular switches, responding to a que by changing properties, such as RNA aptamers that bind fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Sarnie R. Jaffrey. "RNA mimics of green fluorescent protein.” Science 333.6042 (2011): 642-646). It has also been suggested that aptamers may be used as components of targeted siRNA therapeutic delivery systems, for example targeting cell surface proteins (Zhou, Jiehua, and John J. Rossi. "Aptamer-targeted cell-specific RNA interference.” Silence 1.1 (2010): 4).
  • a retron-based gene editing nucleic acid component is modified, e.g., by one or more aptamer(s) designed to improve RNA or DNA component molecule delivery, including delivery across the cellular membrane, to intracellular compartments, or into the nucleus.
  • a structure can include, either in addition to the one or more aptamer(s) or without such one or more aptamer(s), moiety(ies) so as to render the nucleic acid component molecule deliverable, inducible or responsive to a selected effector.
  • the invention accordingly comprehends a reRNA component molecule that responds to normal or pathological physiological conditions, including without limitation pH, hypoxia, oxygen concentration, temperature, protein concentration, enzymatic concentration, lipid structure, light exposure, mechanical disruption (e.g. ultrasound waves), magnetic fields, electric fields, or electromagnetic radiation.
  • Agents that modulate DNA-repair [00743]
  • the retron-based gene editing systems described herein further comprises or encodes a DNA-repair modulating biomolecule, which may further enhance the efficiency of integration of a repair outcome.
  • the DNA-repair modulating biomolecule comprises a Nonhomologous end joining (NHEJ) inhibitor.
  • the DNA-repair modulating biomolecule comprises a homologous directed repair (HDR) promoter.
  • the DNA-repair modulating biomolecule comprises a NHEJ inhibitor and an HDR promoter.
  • the DNA-repair modulating biomolecule enhances or improves more precise genome editing and/or the efficiency of homologous recombination, compared to the otherwise identical embodiment without the DNA-repair modulating biomolecule.
  • HDR promoters and/or NHEJ inhibitors can, in some embodiments, comprise one or more small molecules.
  • recombination enhancers such as small molecules that activate HDR and suppress NHEJ locally at the genomic site of the DNA damage can be tailored in their placement on the engineered systems to further enhance their efficiency.
  • the small molecule recombination enhancers can be synthesized to bear linkers and a functional group, such as maleimide for reacting with a thiol group on a Cys residue of a protein, for chemical conjugation to the engineered systems.
  • linkers and a functional group such as maleimide for reacting with a thiol group on a Cys residue of a protein, for chemical conjugation to the engineered systems.
  • linkers and a functional group such as maleimide for reacting with a thiol group on a Cys residue of a protein
  • Use of commercially available functionalized PEG linkers alkyne, azide, cyclooctyne etc.
  • orthogonal conjugation chemistries can be utilized for the multivalent display.
  • Multivalent display of one or more DNA-repair modulating biomolecule can be effected, including multiple moieties of NHEJ inhibitors, HDR promoters, or a combination thereof. See, for example, “Genomic targeting of epigenetic probes using a chemically tailored Cas9 system” by Liszczak et al., Proc Natl Acad Sci U.S.A.114: 681- 686, 2017 (incorporated herein by reference).
  • multivalent display of small molecule compounds can be achieved through sortase loop proteins used as a scaffold for their display.
  • the DNA-repair modulating biomolecule may comprise an HDR promoter.
  • the HDR promoter may comprise small molecules, such as RSI or analogs thereof.
  • the HDR promoter stimulates RAD51 activity or RAD52 motif protein 1 (RDMl) activity.
  • the HDR promoter comprises Nocodazole, which can result in higher HDR selection.
  • the HDR promoter may be administered prior to the delivery of the retron-based editing systems described herein.
  • the HDR promoter locally enhances HDR without NHEJ inhibition.
  • RAD5l is a protein involved in strand exchange and the search for homology regions during HDR repair.
  • the HDR promoter is phenylbenzamide RSI, identified as a small-molecule RAD51-stimulator (see WO2019/135816 at [0200]-[0204], specifically incorporated herein by reference).
  • the DNA-repair modulating biomolecule comprises C- terminal binding protein interacting protein (CtIP) or a functional fragment or homolog thereof.
  • CtIP is a key protein in early steps of homologous recombination.
  • the CtIP or the functional fragment or homolog thereof can be linked (e.g., fused) to the RT or the sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)), and stimulates transgene integration by HDR.
  • sequence-specific nuclease e.g., a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)
  • the CtIP fragment is a minimal N-terminal fragment of the wild-type CtIP, such as the N-terminal fragment comprising residues 1-296 of the full-length CtIP (the HE for HDR enhancer), as described in Charpentier et al. (Nature Comm., DOI: 10.1038/s41467-018-03475-7, incorporated herein by reference), shown to be sufficient to stimulate HDR.
  • the activity of the fragment depends on CDK phosphorylation sites (e.g., S233, T245, and S276) and the multimerization domain essential for CtIP activity in homologous recombination.
  • the DNA-repair modulating biomolecule comprises a dominant negative 53BP1.
  • the DNA-repair modulating biomolecule comprises a cell cycle-specific degradation tag, such as the degradation domain of the (human) Geminin, and the (murine) CyclinB2.
  • the DNA-repair modulating biomolecule comprises CyclinB2, a member of the B-type cyclins that associate with p34cdc2, and an essential component of the cell cycle regulatory machinery.
  • CRISPR-mediated knock-in efficiency may be increased by promoting the relative increase in Cas9 activity in G2 phase of the cell cycle, when HDR is more active.
  • the degradation domains of the (human) Geminin and (murine) CyclinB2 can be used as either N- or C-terminal fusion to serve as the DNA-repair modulating biomolecule. These domains are known to determine a cell-cycle specific profile of chimeric proteins, namely an increase in their relative concentration in S and G2 compared to G1, high-jacking the conventional CyclinB2 and Geminin degradation pathways. This produces active Geminin-Cas9 and CyclinB2-Cas9 chimeric proteins, which are degraded in a cell-cycle-dependent manner.
  • the DNA-repair modulating biomolecule comprises a Rad family member protein, such as Rad50, Rad51, Rad52, etc., which functions to promote foreign DNA integration into a host chromosome.
  • Rad52 is an important homologous recombinant protein, and its complex with Rad51 plays a key role in HDR, mainly involved in the regulation of foreign DNA in eukaryotes.
  • the DNA-repair modulating biomolecule comprises a RAD52 protein as, e.g., either an N- or a C-terminal fusion.
  • the DNA-repair modulating biomolecule comprises a RAD52 motif protein 1 (RDMl) that functions similarly as RAD52. RDM1 has been shown to be able to repair DSBs caused by DNA replication, prevent G2 or M cell cycle arrest, and improve HDR selection.
  • the DNA-repair modulating biomolecule comprises a dominant negative version of the tumor suppressor p53-binding protein 1 (53BP1).
  • the wild- type protein 53BP1 is a key regulator of the choice between NHEJ and HDR – it is a pro-NHEJ factor which limits HDR by blocking DNA end resection, and also by inhibiting BRCA1 recruitment to DSB sites. It has been shown that global inhibition of 53BP1 by a ubiquitin variant significantly improves Cas9-mediated HDR frequency in non-hematopoietic and hematopoietic cells with single-strand oligonucleotide delivery or double-strand donor in AAV.
  • the dominant negative (DN) version of the 53BP1 comprises the minimal focus forming region, but lacks domains outside this region, e.g., towards the N-terminus and tandem C-terminal BRCT repeats that recruit key effectors involved in NHEJ, such as RIFl-PTIP and EXPAND, respectively.
  • the 53BP1 adapter protein is recruited to specific histone marks at sites of DSBs via this minimal focus forming region, which comprises several conserved domains including an oligomerization domain (OD), a glycine-arginine rich (GAR) motif, a Vietnamese domain, and an adjacent ubiquitin-dependent recruitment (UDR) motif.
  • the Jewish domain mediates interactions with histone H4 dimethylated at K2023.
  • a dominant negative version of 53BP1 suppresses the accumulation of endogenous 53BP1 and downstream NHEJ proteins at sites of DNA damage, while upregulating the recruitment of the BRCA1 HDR protein.
  • DN1S dominant negative version of 53BP1
  • Such a DN version of the 53BP1 can be used as the DNA-repair modulating biomolecule, either as an N- or a C-terminal fusion (such as a Cas9 fusion, to locally inhibit NHEJ at the Cas9-target site defined by its gRNA, while promoting an increase in HDR, and does not globally affect NHEJ, thereby improving cell viability).
  • the DNA-repair modulating biomolecule comprises an NHEJ inhibitor, such as an inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor.
  • NHEJ inhibitor inhibits the NHEJ pathway, enhances HDR, or modulates both.
  • the NHEJ inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor of the NHEJ pathway comprises an SCR7 analog, for example, PK66, PK76, PK409.
  • the NHEJ inhibitor comprises a KU inhibitor, for example, KU5788, and KU0060648.
  • a small molecule NHEJ inhibitor is linked to a polyglycine tripeptide through PEG for sortase-mediated ligation, as described in WO2019/135816, Guimaraes et al., Nat Protoc 8:1787-99, 2013; Theile et al., Nat Protoc 8:1800-7, 2013; and Schmohl et al., Curr Opin Chem Biol 22:122-8, 2014 (all incorporated herein by reference). The same means can also be used for attaching small molecule HDR enhancers to protein.
  • a nucleic acid targeting moiety conjugates based on small molecule inhibitor of DNA-dependent protein kinase (DNA-PK) or heterodimeric Ku (KU70/KU80) can be utilized.
  • KU-0060648 is one potent KU-inhibitors, which can also be functionalized with poly-glycine and used for recombination enhancement.
  • the DNA-repair modulating biomolecule comprises the Tumor Suppressor p53. p53 plays a direct role in DNA repair, including HR regulation, where it affects the extension of new DNA, thereby affecting HDR selection.
  • p53 In vivo, p53 binds to the nuclear matrix and is a rate-limiting factor in repairing DNA structure. p53 regulates DNA repair processes in almost all eukaryotes via transactivation-dependent and -independent pathways, but only the transactivation-independent function of p53 is involved in HR regulation. Wild-type p53 protein can link double stranded breaks to form intact DNA, as well as also playing a role in inhibiting NHEJ. p53 interacts with HR-related proteins, including Rad51, where it controls HR through direct interaction with Rad51.
  • the retron-based gene editing systems may comprise one or more additional accessory proteins having genome modifying functions, including recombinases, invertases, nucleases, polymerases, ligases, deaminases, reverse transcriptases, or epigenetic modifying functions.
  • the accessory proteins may be provided separately.
  • the accessory proteins may be fused to a polypeptide component of the retron-based editing systems, optionally with a linker.
  • the retron-based gene editing systems may further comprise additional polypeptides polypeptides, proteins and/or peptides known in the art.
  • Non-limiting categories of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides, and chimeric polypeptides, receptor, enzymes, hormones, transcription factors, ligands, membrane transporters, structural proteins, nucleases, or a component, variant or fragment (e.g., a biologically active fragment) thereof.
  • peptide generally refers to shorter polypeptides of about 50 amino acids or less.
  • Peptides with only two amino acids may be referred to as “dipeptides.” Peptides with only three amino acids may be referred to as “tripeptides.” Polypeptides generally refer to polypeptides with from about 4 to about 50 amino acids. Peptides may be obtained via any method known to those skilled in the art. In some embodiments, peptides may be expressed in culture. In some embodiments, peptides may be obtained via chemical synthesis (e.g., solid phase peptide synthesis).
  • the RNA payloads may encode a user-programmable DNA binding protein, or a gene editor accessory proteins, such as, but not limited to a deaminases, nucleases, transposases, polymerases, and reverse transcriptases, etc.
  • the RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more encoded products of interest), e.g., the originator constructs and benchmark constructs described herein, may encode a simple protein associated with a non- protein.
  • conjugated proteins include, glycoproteins, hemoglobins, lecithoproteins, nucleoproteins, and phosphoproteins.
  • the RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more encoded products of interest), e.g., the originator constructs and benchmark constructs described herein, may encode a protein that is derived from a simple or conjugated protein by chemical or physical means.
  • derived proteins include denatured proteins and peptides.
  • the polypeptide, protein or peptide may be unmodified.
  • the polypeptide, protein or peptide may be modified.
  • Types of modifications include, but are not limited to, phosphorylation, glycosylation, acetylation, ubiquitylation/sumoylation, methylation, palmitoylation, quinone, amidation, myristoylation, pyrrolidone carboxylic acid, hydroxylation, phosphopantetheine, prenylation, GPI anchoring, oxidation, ADP-ribosylation, sulfation, S-nitrosylation, citrullination, nitration, gamma-carboxyglutamic acid, formylation, hypusine, topaquinone (TPQ), bromination, lysine topaquinone (LTQ), tryptophan tryptophylquinone (TTQ), iodination, and cysteine tryptophylquinone (CTQ).
  • phosphorylation glycosylation, acetylation, ubiquitylation/sumoylation, methylation, palmitoylation, quin
  • the polypeptide, protein or peptide may be modified by a post-transcriptional modification which can affect its structure, subcellular localization, and/or function.
  • the polypeptide, protein or peptide may be modified using phosphorylation. Phosphorylation, or the addition of a phosphate group to serine, threonine, or tyrosine residues, is one of most common forms of protein modification. Protein phosphorylation plays an important role in fine tuning the signal in the intracellular signaling cascades.
  • the polypeptide, protein or peptide may be modified using ubiquitination which is the covalent attachment of ubiquitin to target proteins.
  • the polypeptide, protein or peptide may be modified using acetylation and methylation which can play a role in regulating gene expression.
  • the acetylation and methylation could mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin) which could have an impact on mediating gene silencing.
  • the polypeptide, protein or peptide may be modified using glycosylation.
  • Glycosylation is the attachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins and plays a role in biological processes including, but not limited to, embryonic development, cell division, and regulation of protein structure.
  • the two main types of protein glycosylation are N-glycosylation and O-glycosylation.
  • N-glycosylation the glycan is attached to an asparagine
  • O-glycosylation the glycan is attached to a serine or threonine.
  • the polypeptide, protein or peptide may be modified using sumoylation.
  • RNA payloads e.g., linear and/or circular mRNA payloads encoding one or more encoded products of interest
  • the originator constructs and benchmark constructs described herein may encode a therapeutic protein, such as those exemplified below.
  • the RNA payloads may encode a gene editing system, such as those exemplified herein.
  • a “nucleobase editing system” is a protein, DNA, or RNA composition capable of making edits, modifications or alterations to one or more targeted genes of interest.
  • RNA payloads e.g., linear and/or circular mRNA payloads encoding one or more encoded products of interest
  • a retron-based editing system may be configured as an inducible gene editing system.
  • the inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy.
  • the form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy.
  • the retron-based editing system or component thereof may include a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner.
  • LITE Light Inducible Transcriptional Effector
  • the components of a light may include a retron-editing system component, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain.
  • inducible DNA binding proteins and methods for their use are provided in US Provisional Application Nos.61/736,465 and US 61/721,283, and International Patent Publication No. WO 2014/018423 A2 which is hereby incorporated by reference in its entirety.
  • WO 2014/018423 A2 is hereby incorporated by reference in its entirety.
  • the self-inactivating system includes additional RNA (e.g., nucleic acid component molecule) that targets the coding sequence for the Cas12a polypeptide itself or that targets one or more non-coding nucleic acid component molecule target sequences complementary to unique sequences present in one or more of the following: (a) within the promoter driving expression of the non-coding RNA elements, (b) within the promoter driving expression of the Cas12a polypeptide gene, (c) within 100 bp of the ATG translational start codon in the Cas12a polypeptide coding sequence, (d) within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in the AAV genome.
  • RNA e.g., nucleic acid component molecule
  • a single nucleic acid component molecule is provided that is capable of hybridization to a sequence downstream of a Cas12a polypeptide start codon, whereby after a period of time there is a loss of the Cas12a polypeptide expression.
  • one or more nucleic acid component molecule(s) are provided that are capable of hybridization to one or more coding or non-coding regions of the polynucleotide encoding the system, whereby after a period of time there is a inactivation of one or more, or in some cases all, of the system.
  • the cell may comprise a plurality of complexes, wherein a first subset of complexes comprise a first nucleic acid component molecule capable of targeting a genomic locus or loci to be edited, and a second subset of complexes comprise at least one second nucleic acid component molecule capable of targeting the polynucleotide encoding the system, wherein the first subset of complexes mediate editing of the targeted genomic locus or loci and the second subset of complexes eventually inactivate the system, thereby inactivating further expression in the cell.
  • the various coding sequences can be included on a single vector or on multiple vectors. For instance, it is possible to encode the enzyme on one vector and the various RNA sequences on another vector, or to encode the enzyme and one nucleic acid component molecule on one vector, and the remaining nucleic acid component molecule on another vector, or any other permutation. In general, a system using a total of one or two different vectors is preferred.
  • H. Vectors for Expression of Engineered Retrons [00793] Delivery of an engineered retron to a cell can generally be accomplished with or without vectors.
  • Delivery of the ncRNA encoded by the engineered retron generally does not require a vector used to produce the ncRNA from the engineered retron.
  • the ncRNA can be packaged directly into a delivery vehicle such as a lipid nanoparticle and delivered into a host cell, as described in other sections.
  • the engineered retrons may be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants (e.g., monocotyledonous and dicotyledonous plants); and animals (e.g., vertebrates and invertebrates).
  • Examples of animals that may be transfected with an engineered retron include, without limitation, vertebrates such as fish, birds, mammals (e.g., human and non-human primates, farm animals, pets, and laboratory animals), reptiles, and amphibians.
  • the engineered retrons can be introduced into a single cell or a population of cells. Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be transfected with the engineered retrons.
  • the engineered retrons can be introduced into cellular fragments, cell components, or organelles (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae).
  • organelles e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae.
  • Cells may be cultured or expanded after transfection with the engineered retron.
  • Methods of introducing nucleic acids into a host cell are well known in the art.
  • Commonly used methods include chemically induced transformation, typically using divalent cations (e.g., CaCl 2 ), dextran-mediated transfection, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of the nucleic acids comprising engineered retrons into nuclei.
  • divalent cations e.g., CaCl 2
  • the plant cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional methods.
  • Plant material that may be transformed with the engineered retrons described herein includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the genetic modification introduced by the engineered retron.
  • the engineered retrons described herein may be used to produce transgenic plants with desired phenotypes, including but not limited to, increased disease resistance (e.g., increased viral, bacterial of fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil composition, and color.
  • the retron msr gene, msd gene, and/or ret gene are expressed in vitro from a vector, such as in an in vitro transcription system.
  • the resulting ncRNA or msDNA can be isolated before being packaged and/or formulated for direct delivery into a host cell.
  • the isolated ncRNA or msDNA can be packaged/formulated in a delivery vehicle such as lipid nanoparticles as described in other sections.
  • a delivery vehicle such as lipid nanoparticles as described in other sections.
  • the retron msr gene, msd gene, and/or ret gene are expressed in vivo from a vector within a cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • the retron msr gene, msd gene, and/or ret gene, and any other components of the retron-based genome editing systems described herein may be expressed in vivo from RNA delivered to the cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • Vectors and/or nucleic acid molecules encoding the recombinant retron-based genome editing system or components thereof can include control elements operably linked to the retron sequences, which allow for the production of msDNA either in vitro, or in vivo in the subject species.
  • the retron msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retron reverse transcriptase and/or the msDNA product.
  • heterologous sequences encoding desired products of interest may be inserted in the msr gene and/or msd gene.
  • desired products of interest e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording
  • desired products of interest e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording
  • desired products of interest e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording
  • Any eukaryotic, archaeal, or prokaryotic cell capable of being transfected with a vector or retron delivery system comprising the engineered retron sequences, may be used to produce the msDNA in vivo.
  • the transfected cell can be assayed either through phenotypic changes that occur due to the introduced sequences or by direct DNA sequencing.
  • the engineered retron is produced by a vector system comprising one or more vectors.
  • the msr gene, the msd gene, and/or the ret gene may be provided by the same vector (i.e., cis arrangement of all such retron elements), wherein the vector comprises a promoter operably linked to the msr gene and/or the msd gene.
  • the promoter is further operably linked to the ret gene.
  • the vector further comprises a second promoter operably linked to the ret gene.
  • the ret gene may be provided by a second vector that does not include the msr gene and/or the msd gene (i.e., trans arrangement of msr-msd and ret).
  • the msr gene, the msd gene, and the ret gene are each provided by different vectors (i.e., trans arrangement of all retron elements).
  • Numerous vectors are available for use in the vector or vector system, including but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • the nucleic acid comprising an engineered retron sequence is under transcriptional control of a promoter.
  • the promoter is competent for initiating transcription of an operably linked coding sequence by a RNA polymerase I, II, or III.
  • Exemplary promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U. S. Patent Nos. 5,168,062 and 5,385,839, incorporated herein by reference in their entireties), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others.
  • Other nonviral promoters such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • Exemplary promoters for plant cell expression include the CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171); the ubiquitin promoter (Christensen et al., 1989, Plant Mol. Biol.12:619-632; and Christensen et al., 1992, Plant Mol. Biol.18:675-689); the pEMU promoter (Last et al., 1991, Theor. Appl. Genet.81:581-588); and the MAS promoter (Velten et al., 1984, EMBO J.
  • the retron-based vectors may also comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.
  • tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • promoters can be obtained from or incorporated into commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra.
  • one or more enhancer elements is/are used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBOJ (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci.
  • LTR long terminal repeat
  • an expression vector for expressing an engineered retron including the msr gene, msd gene, and/or ret gene comprises a promoter operably linked to a polynucleotide encoding the msr gene, msd gene, and/or ret gene.
  • the vector or vector system also comprises a transcription terminator/polyadenylation signal.
  • sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Patent No.5,122,458).
  • 5 ⁇ - UTR sequences can be placed adjacent to the coding sequence to further enhance the expression.
  • Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • IRES internal ribosome entry site
  • IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al.. Virol. (1989) 63:1651-1660).
  • EMCV encephalomyocarditis virus
  • the polio leader sequence the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301)).
  • an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J Biol. Chem. (2004) 279(51):3389-33971) and the like.
  • IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol.24( 17): 7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad Sci. U.S.A.105(12):4733-4738, Stein et al. (1998) Mol. Cell.
  • An IRES sequence may be included in a vector, for example, to express multiple bacteriophage recombination proteins for recombineering or an RNA-guided nuclease (e.g., Cas9) for HDR in combination with a retron reverse transcriptase from an expression cassette.
  • a polynucleotide encoding a viral self-cleaving 2A peptide, such as a T2A peptide can be used to allow production of multiple protein products (e.g., Cas9, bacteriophage recombination proteins, retron reverse transcriptase) from a single vector or a single transcription unit under one promoter.
  • One or more 2A linker peptides can be inserted between the coding sequences in the multicistronic construct.
  • the 2A peptide which is self- cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels.
  • 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Jhosea asigna virus and porcine teschovirus-1. See, e.g., Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol.6:40, Provost et al.
  • the expression construct comprises a plasmid suitable for transforming a bacterial host.
  • Numerous bacterial expression vectors are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice.
  • Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31
  • Bacterial plasmids may contain antibiotic selection markers (e.g., ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), a lacZ gene (b-galactosidase produces blue pigment from x-gal substrate), fluorescent markers (e.g., GFP.
  • the expression construct comprises a plasmid suitable for transforming a yeast cell.
  • Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), antibiotic selection markers (e.g., kanamycin resistance), fluorescent markers (e.g., mCherry), or other markers for selection of transformed yeast cells.
  • ORI yeast-specific origin of replication
  • nutritional selection markers e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+
  • antibiotic selection markers e.g., kanamycin resistance
  • fluorescent markers e.g., mCherry
  • the yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E coif) and yeast cells.
  • yeast plasmids include yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.
  • Yip yeast integrating plasmids
  • ARS autonomously replicating sequence
  • YCp yeast centromere plasmids
  • the expression construct does not comprise a plasmid suitable for transforming a yeast cell.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Wamock et al. (2011) Methods Mol. Biol.737:1-25; Walther et al.
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat.
  • Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).
  • a number of adenoviral vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor LaboratoryPress); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N.
  • Another vector system useful for delivering nucleic acids encoding the engineered retrons is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct.14, 1997, herein incorporated by reference).
  • viral vectors include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing a nucleic acid molecule of interest can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • avipoxviruses such as the fowlpox and canarypox viruses, can also be used to deliver the nucleic acid molecules of interest.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat.
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol.77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the nucleic acids of interest (e.g., engineered retron) in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the nucleic acid of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA.
  • RNA messenger RNA
  • Elroy-Stein and Moss Proc. Natl. Acad. Sci. USA (1990) 87:6743- 6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more templates. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • T7 systems and their use for transforming cells see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S.
  • Insect cell expression systems such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, D.W. Murhammer ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA).
  • Plant expression systems can also be used for transforming plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; andhackland et al., Arch. Virol. (1994) 139:1-22.
  • the expression construct or the ncRNA must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.
  • Several non-viral methods for the transfer of expression constructs into cultured cells also are contemplated. These include the use of calcium phosphate precipitation, DEAE- dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (see, e.g., Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol.7:2745-2752; Rippe et al. (1990) Mol. Cell Biol.
  • nucleic acid comprising the engineered retron sequence may be positioned and expressed at different sites.
  • the nucleic acid comprising the engineered retron sequence may be stably integrated into the genome of the cell.
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. [00841] In some embodiments, the expression construct may simply consist of naked recombinant DNA or plasmids comprising the engineered retron.
  • Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533
  • Benvenisty & Neshif Proc. Natl. Acad. Sci. USA (1986) 83:9551-9555
  • direct intraperitoneal injection of calcium phosphate- precipitated plasmids results in expression of the transfected genes.
  • DNA encoding an engineered retron of interest may also be transferred in a similar manner in vivo and express retron products.
  • a naked DNA expression construct may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al. (1987) Nature 327:70-73).
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572).
  • the microprojectiles may consist of biologically inert substances, such as tungsten or gold beads.
  • the expression construct may be delivered using liposomes.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al.
  • the liposome may be complexed or employed in conjunction with nuclear non- histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem.266(6):3361-3364).
  • HMG-I nuclear non- histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-I.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • Other expression constructs which can be employed to deliver a nucleic acid into cells are receptor-mediated delivery vehicles.
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin (see, e.g., Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci.
  • the delivery vehicle may comprise a ligand and a liposome.
  • Nicolau et al. Methods Enzymol.
  • the promoters that may be used in the retron delivery systems described herein may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters.
  • Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing.
  • the promoter may be a CMV promoter.
  • the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue.
  • Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • B29 promoter include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-
  • the disclosure provides vectors for transferring and/or expressing said retron-based gene editing systems, e.g., under in vitro, ex vivo, and in vivo conditions.
  • the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles of the retron-based gene editing systems described herein.
  • the retron-based gene editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., guide RNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., guide RNA and mRNA delivered by LNPs
  • a mixture of DNA and RNA e.g., protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • RNP ribonucleoprotein
  • the retron-based gene editing systems and/or components thereof can be delivered by any known delivery system such as those described above, including (a) without vectors (e.g., electroporation), (b) viral delivery systems and (c) non-viral delivery systems.
  • Viral delivery systems include expression vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • Non-viral delivery systems include without limitation lipid particles (e.g.
  • Lipid nanoparticles (LNPs)), non-lipid nanoparticles, exosomes, liposomes, micelles, viral particles, stable nucleic-acid-lipid particles (SNALPs), lipoplexes/polyplexes, DNA nanoclews, Gold nanoparticles, iTOP, Streptolysin O (SLO), multifunctional envelope- type nanodevice (MEND), lipid-coated mesoporous silica particles, inorganic nanoparticles, and polymeric delivery technology (e.g., polymer-based particles).
  • SNALPs stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-
  • the engineered retron-based gene editing systems may be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants (e.g., monocotyledonous and dicotyledonous plants); and animals (e.g., vertebrates and invertebrates).
  • animals e.g., vertebrates and invertebrates.
  • animals that may be transfected with an engineered retron-based gene editing systems include, without limitation, vertebrates such as fish, birds, mammals (e.g., human and non-human primates, farm animals, pets, and laboratory animals), reptiles, and amphibians.
  • the engineered retron-based gene editing systems can be introduced into a single cell or a population of cells.
  • Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be transfected with the engineered retron-based gene editing systems.
  • the engineered retron-based gene editing systems can be introduced into cellular fragments, cell components, or organelles (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae).
  • organelles e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae.
  • Cells may be cultured or expanded after transfection with the engineered retron- based gene editing systems.
  • Methods of introducing nucleic acids into a host cell are well known in the art.
  • Commonly used methods include chemically induced transformation, typically using divalent cations (e.g., CaCl 2 ), dextran-mediated transfection, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of the nucleic acids comprising Cas12a editing systems into nuclei.
  • divalent cations e.g., CaCl 2
  • Plant cells may also be targeted by the retron-based gene editing systems (or components thereof) disclosed herein.
  • Methods for genetic transformation of plant cells are known in the art and include those set forth in US2022/0145296, and U.S. Pat. Nos.8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference in its entirety. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett.7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al.
  • Plant material that may be transformed with the retron-based gene editing systems (or components thereof) described herein includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
  • Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the genetic modification introduced by the retron- based gene editing systems. Further provided is a processed plant product or byproduct that retains the genetic modification introduced by the retron-based gene editing systems.
  • the retron-based gene editing systems described herein may be used to produce transgenic plants with desired phenotypes, including but not limited to, increased disease resistance (e.g., increased viral, bacterial of fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil composition, and color.
  • the retron msr gene, msd gene, and/or ret gene can be expressed in vitro from a vector, such as in an in vitro transcription system.
  • the resulting ncRNA or msDNA can be isolated before being packaged and/or formulated for direct delivery into a host cell.
  • the isolated ncRNA or msDNA can be packaged/formulated in a delivery vehicle such as lipid nanoparticles as described in other sections.
  • the retron msr gene, msd gene, and/or ret gene are expressed in vivo from a vector within a cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • the retron msr gene, msd gene, and/or ret gene, and any other components of the retron-based genome editing systems described herein may be expressed in vivo from RNA delivered to the cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • Vectors and/or nucleic acid molecules encoding the recombinant retron-based genome editing system or components thereof can include control elements operably linked to the retron sequences, which allow for the production of msDNA either in vitro, or in vivo in the subject species.
  • the retron msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retron reverse transcriptase and/or the msDNA product.
  • heterologous sequences encoding desired products of interest e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording
  • the retron-based gene editing systems are produced by a vector system comprising one or more vectors.
  • vectors are available for use in the vector or vector system, including but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • All-RNA format [00867]
  • the retron-based gene editing systems (or components thereof) disclosed herein may be delivered in an “all-RNA” format.
  • the term “all-RNA” format refers to the fact that each of the components of a retron editing system (e.g., the retron RT, the programmable nuclease, the sgRNA, and the ncRNA) are delivered and/or administered as RNA (e.g., coding RNA or non-coding RNA).
  • the RNA components may be delivered to cells and/or tissues by direct means, such as electroporation or transfection.
  • the RNA components may be delivered to cells and/or tissues by way of a delivery vehicle, such as an LNP or liposome.
  • the retron editing systems described herein may comprise a coding RNA (e.g., linear or circular mRNA) that encodes a retron reverse transcriptase (e.g., any RT from Table X or Table A), a coding RNA (e.g., linear or circular mRNA) that encodes a programmable nuclease (e.g., a Cas9, Cas12a, or TnpB nuclease), a retron ncRNA (e.g., a ncRNA from Table B), and a guide RNA for targeting the programmable nuclease to a particular desired target sequence.
  • a coding RNA e.g., linear or circular mRNA
  • a coding RNA e.g., linear or circular mRNA
  • a programmable nuclease e.g., a Cas9, Cas12a, or TnpB nuclease
  • RT and nuclease components may be encoded on the same coding RNA molecule.
  • the proteins may also be expressed from separate coding RNA molecules.
  • the RT and the nuclease components can be fused together as a singular fusion polypeptide having an RT domain and a nuclease domain optionally joined by a linker.
  • the ncRNA and the guide RNA may be fused together as a single RNA molecule.
  • the guide RNA may be located at the 5’ end of the ncRNA. In other embodiments, the guide RNA may be located at the 3’ end of the ncRNA.
  • the ncRNA may comprise a guide RNA at both the 3’ and the 5’ ends of the ncRNA.
  • the ncRNA and the guide RNA may be separate molecules, i.e., delivered separately.
  • the retron editing system may include both a ncRNA- guide RNA fusion and an additional guide RNA provided as a separate molecule.
  • the different RNA components of the all-RNA retron editing system can be combined and administered (e.g., directly or within a delivery vehicle) in different ratios. In some embodiments, the ratios of such RNA components or species can be expressed as molar ratios.
  • the molar ratio of RT coding RNA to nuclease coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of nuclease coding RNA to RT coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of ncRNA or ncRNA-guide RNA fusion to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of separate guide RNA to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of ncRNA to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of separate guide RNA to ncRNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA (e.g., encoding RT and/or nuclease) to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a retron RT to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a programmable nuclease to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a retron RT or a nuclease to a separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a separate guide RNA to a coding RNA encoding a retron RT or a nuclease can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00885] In certain embodiments, the amount of ncRNA-sgRNA relative to RT mRNA is augmented.
  • the RT mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • an RT-Cas9 (or Cas9-RT) fusion is encoded by an mRNA.
  • the RT-Cas9 mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • multiple genetic loci are targeted hence the ncRNA-sgRNA includes a mixture of ncRNA-sgRNA species and the same ratios and ranges are applicable.
  • the retron-based gene editing systems described herein may be delivered in viral vectors.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • the nucleic acid comprising an retron-based gene editing system (or component thereof) is under transcriptional control of a promoter.
  • the promoter is competent for initiating transcription of an operably linked coding sequence by a RNA polymerase I, II, or III.
  • Exemplary promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U.S. Patent Nos. 5,168,062 and 5,385,839, incorporated herein by reference in their entireties), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others.
  • Other nonviral promoters such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • Exemplary promoters for plant cell expression include the CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171); the ubiquitin promoter (Christensen et al., 1989, Plant Mol. Biol.12:619-632; and Christensen et al., 1992, Plant Mol. Biol.18:675-689); the pEMU promoter (Last et al., 1991, Theor. Appl. Genet.81:581-588); and the MAS promoter (Velten et al., 1984, EMBO J.
  • the retron-based vectors may also comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.
  • tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • promoters can be obtained from or incorporated into commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra.
  • one or more enhancer elements is/are used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBOJ (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci.
  • LTR long terminal repeat
  • an expression vector for expressing a retron-based gene editing system (or component thereof) comprises a promoter operably linked to a polynucleotide encoding the components.
  • the components of the retron-based gene editing system may be configured as individual gene transcripts or as fused constructs.
  • the nuclease component may be fused with the reverse transcriptase component.
  • the ncRNA component may be fused with the guide RNA component.
  • the nuclease component may be fused with the reverse transcriptase component, but the guide RNA and the ncRNA are separate.
  • the guide RNA and ncRNA components may be fused, but the reverse transcripase and nuclease components are separately provided. Any functional combinations of fused components and non-fused components are contemplated.
  • the vector or vector system also comprises a transcription terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S.
  • UTR sequences can be placed adjacent to the coding sequence to further enhance the expression.
  • Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • IRES permits the translation of one or more open reading frames from a vector.
  • the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm.
  • IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al.. Virol. (1989) 63:1651-1660).
  • EMCV encephalomyocarditis virus
  • the polio leader sequence the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301)).
  • an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J Biol. Chem. (2004) 279(51):3389-33971) and the like.
  • IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol.24( 17): 7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad Sci. U.S.A.105(12):4733-4738, Stein et al. (1998) Mol. Cell.
  • An IRES sequence may be included in a vector, for example, to express multiple bacteriophage recombination proteins for recombineering or an RNA-guided nuclease (e.g., Cas9) for HDR in combination with a retron reverse transcriptase from an expression cassette.
  • a polynucleotide encoding a viral self-cleaving 2A peptide, such as a T2A peptide can be used to allow production of multiple protein products (e.g., Cas9, bacteriophage recombination proteins, retron reverse transcriptase) from a single vector or a single transcription unit under one promoter.
  • One or more 2A linker peptides can be inserted between the coding sequences in the multicistronic construct.
  • the 2A peptide which is self- cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels.
  • 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Jhosea asigna virus and porcine teschovirus-1. See, e.g., Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol.6:40, Provost et al.
  • the expression construct comprises a plasmid suitable for transforming a bacterial host.
  • Numerous bacterial expression vectors are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice.
  • Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31
  • Bacterial plasmids may contain antibiotic selection markers (e.g., ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), a lacZ gene (b-galactosidase produces blue pigment from x-gal substrate), fluorescent markers (e.g., GFP.
  • the expression construct comprises a plasmid suitable for transforming a yeast cell.
  • Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), antibiotic selection markers (e.g., kanamycin resistance), fluorescent markers (e.g., mCherry), or other markers for selection of transformed yeast cells.
  • ORI yeast-specific origin of replication
  • nutritional selection markers e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+
  • antibiotic selection markers e.g., kanamycin resistance
  • fluorescent markers e.g., mCherry
  • the yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E coif) and yeast cells.
  • yeast plasmids include yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.
  • Yip yeast integrating plasmids
  • ARS autonomously replicating sequence
  • YCp yeast centromere plasmids
  • the expression construct does not comprise a plasmid suitable for transforming a yeast cell.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Wamock et al. (2011) Methods Mol. Biol.737:1-25; Walther et al.
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat.
  • Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).
  • a number of adenoviral vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor LaboratoryPress); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N.
  • Another vector system useful for delivering nucleic acids encoding the Cas12a editing system components is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct.14, 1997, herein incorporated by reference).
  • viral vectors include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing a nucleic acid molecule of interest can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • avipoxviruses such as the fowlpox and canarypox viruses, can also be used to deliver the nucleic acid molecules of interest.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat.
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol.77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the nucleic acids of interest (e.g., Cas12a editing system) in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the nucleic acid of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA.
  • RNA messenger RNA
  • Elroy-Stein and Moss Proc. Natl. Acad. Sci. USA (1990) 87:6743- 6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more templates. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • T7 systems and their use for transforming cells see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S.
  • Insect cell expression systems such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, D.W. Murhammer ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA).
  • Plant expression systems can also be used for transforming plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; andhackland et al., Arch. Virol. (1994) 139:1-22.
  • the expression constructs and/or RNA components must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • Non-viral delivery methods [00916] Several non-viral methods for the transfer of expression constructs are available for delivering the retron-based editing systems or components thereof into cells also are contemplated. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (see, e.g., Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol.
  • nucleic acid molecules encoding the retron-based editing systems or components thereof may be stably integrated into the genome of the cell.
  • nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
  • nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • expression constructs encoding the retron-based editing systems or components thereof may simply consist of naked recombinant DNA or plasmids comprising nucleotide sequences encoding said retron-based editing systems or components thereof. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • DNA expression constructs encoding the retron- based editing systems or components thereof may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al.
  • Liposomes [00920] In a further embodiment, constructs encoding the retron-based editing systems or components thereof may be delivered using liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium.
  • Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh & Bachhawat (1991) Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104). Also contemplated is the use of lipofectamine-DNA complexes.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al. (1989) Science 243:375-378).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non- histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem.266(6):3361-3364).
  • HMG-I nuclear non- histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-I.
  • Liposomes can be made from several different types of lipids, e.g., phospholipids.
  • a liposome may comprise natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3 - phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof.
  • DSPC l,2-distearoryl-sn-glycero-3 - phosphatidyl choline
  • sphingomyelin sphingomyelin
  • egg phosphatidylcholines monosialoganglioside, or any combination thereof.
  • Several other additives may be added to liposomes in order to modify their structure and properties.
  • liposomes may further comprise cholesterol, sphingomyelin, and/or 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), e.g., to increase stability and/or to prevent the leakage of the liposomal inner cargo.
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • the liposome comprises a transport polymer, which may optionally be branched, comprising at least 10 amino acids and a ratio of histidine to non- histidine amino acids greater than 1.5 and less than 10.
  • the branched transport polymer can comprise one or more backbones, one or more terminal branches, and optionally, one or more non-terminal branches. See, U.S.
  • the transposrt polymer is a Histidine-Lysine co-polymer (HKP) used to package and deliver mRNA and other cargos. See, U.S. Patent Nos., 7,163,695, and 7,772,201 , incorporated herein by reference in their entireties, [00926]
  • the lipid particles may be stable nucleic acid lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof.
  • DLinDMA ionizable lipid
  • PEG diffusible polyethylene glycol
  • SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane.
  • SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG- eDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).
  • the retron-based editing systems or components thereof may be encapsulated by delivery vehicles that comprise polymer-based particles (e.g., nanoparticles).
  • the polymer-based particles may mimic a viral mechanism of membrane fusion.
  • the polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or snucleic acid component, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment.
  • the low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing.
  • the polymer-based particles may comprise alkylated and carboxyalkylated branched polyethylenimine.
  • the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA.
  • Example methods of delivering the systems and compositions herein include those described in Bawage SS et al., Synthetic mRNA expressed Cast 3a mitigates RNA virus infections, biorxiv.org/content/10.1101/370460vl.
  • the delivery vehicles may comprise exosomes.
  • Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs).
  • exosomes examples include those described in Schroeder A, et al., J Intern Med. 2010 Jan;267(l):9-21; El-Andaloussi S, et al., Nat Protoc.2012 Dec;7(12):2112-26; Uno Y, et al., Hum [00929] Gene Ther.2011 Jun;22(6):711-9; Zou W, et al., Hum Gene Then 2011 Apr;22(4):465-75.
  • Exemplary exosomes can be generated from 293F cells, with mRNA-loaded exosomes driving higher mRNA expression than mRNA loaded LNPs in some instances. See, e.g. J. Biol. Chem.
  • the exosome may form a complex (e.g., by binding directly or indirectly) to one or more components of the cargo.
  • a molecule of an exosome may be fused with first adapter protein and a component of the cargo may be fused with a second adapter protein.
  • the first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome. Examples of such exosomes include those described in Ye Y, et al., Biomater Sci.2020 Apr 28. doi: 10.1039/d0bm00427h.
  • Receptor-mediated delivery [00931] Other expression constructs encoding the retron-based editing systems or components thereof are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu (1993) Adv. Drug Delivery Rev.12:159- 167).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer.
  • a synthetic neoglycoprotein which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al. (1993) FASEB J.7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci.
  • delivery vehicle comprising one or more expression constructs encoding the retron-based editing systems or components thereof may comprise a ligand and a liposome.
  • Nicolau et al. Methods Enzymol. (1987) 149:157-176) employed lactosy 1-ceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell by any number of receptor-ligand systems with or without liposomes.
  • antibodies to surface antigens on cells can similarly be used as targeting moieties.
  • the promoters that may be used in the retron-based editing systems or components thereof described herein may be constitutive, inducible, or tissue- specific. In some embodiments, the promoters may be a constitutive promoters.
  • Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing.
  • the promoter may be a CMV promoter.
  • the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue.
  • Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • LNPs Lipid nanoparticles
  • the retron-based editing systems or components thereof e.g., linear and circular mRNAs; nucleobase editing systems and/or components thereof
  • LNPs lipid nanoparticles
  • compositions and/or formulations comprising RNA-encapsulated LNPs.
  • LNPs that may be used as the payload delivery vehicles contemplated herein, as well as the various ionizable lipids, structural lipids, PEGylated lipids, and phospholipids that may be used to make the herein LNPs for delivery payloads to cells.
  • the present disclosure further provides delivery systems for delivery of a therapeutic payload (e.g., the RNA payloads described herein which may encode a polypeptide of interest, e.g., a nucleobase editing system or a therapeutic protein) disclosed herein.
  • a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.
  • LNP lipid nanoparticle
  • an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid.

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Abstract

Des rétrons modifiés et des méthodes d'utilisation permettant de modifier le génome d'une cellule hôte (par exemple, cellule de mammifère) par administration du rétron modifié ou de l'ARNnc codé in vitro ou in vivo à la cellule hôte (par exemple, cellule de mammifère) sont divulgués.<i />
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WO2024192291A1 (fr) * 2023-03-15 2024-09-19 Renagade Therapeutics Management Inc. Administration de systèmes d'édition de gènes et leurs procédés d'utilisation
CN119530107A (zh) * 2025-01-23 2025-02-28 中国海洋大学 一种产tda的海洋生物被膜细菌、益生菌制剂、饲料及应用
WO2025081042A1 (fr) 2023-10-12 2025-04-17 Renagade Therapeutics Management Inc. Système d'édition de précision basé sur un modèle de nickase-rétron et méthodes d'utilisation
WO2025128871A2 (fr) 2023-12-13 2025-06-19 Renagade Therapeutics Management Inc. Nanoparticules lipidiques comprenant des molécules d'arn codant destinées à être utilisées dans l'édition de gènes et en tant que vaccins et agents thérapeutiques

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