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KR-102963335-B1 - Improved Homology-Dependent Repair Genome Editing

KR102963335B1KR 102963335 B1KR102963335 B1KR 102963335B1KR-102963335-B1

Abstract

Eukaryotic cells and related reagents, systems, methods, and compositions for increasing the frequency of homology-designated repair (HDR) of target edit sites using genome editing molecules are provided.

Inventors

  • 세르막, 토마스

Assignees

  • 이나리 아그리컬쳐 테크놀로지, 인크.

Dates

Publication Date
20260511
Application Date
20200624
Priority Date
20190625

Claims (20)

  1. A method for increasing homology-designated repair (HDR)-mediated genomic modifications of target edit sites in eukaryotic cell genomes, comprising the following: A step of providing a genome-editing molecule and an HDR promoter to a eukaryotic cell, wherein the genome-editing molecule and the HDR promoter provide modification of a target edit site of a eukaryotic cell genome using a donor template polynucleotide by HDR at an increased frequency compared to a control group, wherein the genome-editing molecule comprises (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding a sequence-specific endonuclease that cleaves a DNA sequence within the target edit site; and (ii) a donor template DNA molecule having homology to the target edit site; and wherein the HDR promoter comprises a single-stranded DNA annealing protein (SSAP), an exonuclease capable of converting a double-stranded DNA substrate at least partially into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB).
  2. In claim 1, the sequence-specific endonuclease (a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and a guide RNA or a polynucleotide encoding a guide RNA, and/or; (b) comprising zinc-finger nucleases (ZFN), transcriptional activator-like effector nucleases (TAL-effector nucleases), Argonautes, meganucleases, or engineered meganucleases; (c) comprising an RNA-guided nuclease, wherein the target editing site comprises a sequence that is complementary to the PAM sequence and the guide RNA and is located immediately adjacent to the protospacer adjacent motif (PAM) sequence; (d) providing a 5' overhang to the target edit area after cutting. method.
  3. A method according to claim 1, comprising one or more sequence-specific endonucleases or sequence-specific endonucleases and guide RNA, wherein the genome editing molecule cuts a single DNA strand at two distinct DNA sequences within a target editing site.
  4. In paragraph 3, (a) The sequence-specific endonuclease comprises at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof; (b) the sequence-specific endonuclease comprises Cas9 and/or Cas12a, and the guide RNA molecule has at least one base mismatch with respect to the DNA sequence within the target editing site; (c) The exonuclease comprises a T7 phage exonuclease, Escherichia coli ( E. coli ) exonuclease III, an associated protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 143 or 144. method.
  5. In claim 1, the donor template DNA molecule (a) provided as a circular DNA vector, on a Geminivirus replicon or as a linear DNA fragment; (b) A copy of the endonuclease recognition sequence is adjacent method.
  6. In paragraph 1, the above SSAP (a) Provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules; (b) containing RecT/Redβ-, ERF-, or RAD52- and proteins, wherein (i) the above RecT/Redβ- and protein comprises the Rac bacterial prophage RecT protein, bacteriophage λ beta protein, bacteriophage SPP1 35 protein, an associated protein having equivalent SSAP activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 1, 2, or 3; (ii) The above ERF- and protein comprises a bacteriophage P22 ERF protein, a functionally related protein, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 4; (iii) the above RAD52- and protein comprising a Saccharomyces cerevisiae Rad52 protein, a Schizosaccharomyces pombe Rad22 protein, a Kluyveromyces lactis Rad52 protein, a functionally related protein, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 5, 6, or 7 method.
  7. (a) Eukaryotic cell; (b) an HDR promoter comprising a single-stranded DNA annealing protein (SSAP), an exonuclease capable of converting a double-stranded DNA substrate at least partially into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); and (c) Genome editing molecule(s) comprising at least one sequence-specific endonuclease that cleaves a DNA sequence within a target editing site, or at least one polynucleotide encoding a sequence-specific endonuclease, and a donor template DNA molecule having homology to the target editing site. As a system for increasing High Dynamic Range (HDR)-mediated genomic modifications of target edit sites in eukaryotic cell genomes including, The above eukaryotic cell is associated with and/or contacts with an effective amount of HDR promoter and genome editing molecule(s) and/or contains them. System.
  8. In claim 7, the genome editing molecule (a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease, and a guide RNA or a polynucleotide encoding a guide RNA, and/or; (b) comprising zinc-finger nucleases (ZFN), transcriptional activator-like effector nucleases (TAL-effector nucleases), argonauts, meganucleases, or engineered meganucleases; (c) comprising one or more sequence-specific endonucleases or sequence-specific endonucleases and guide RNAs that cleave a single DNA strand at two distinct DNA sequences within a target editing site, wherein the sequence-specific endonucleases (i) comprising at least one Cas9 nikkaze, Cas12a nikkaze, Cas12i, zinc finger nikkaze, TALE nikkaze, or a combination thereof; (ii) comprising Cas9 and/or Cas12a, wherein the guide RNA molecule has at least one base mismatch with respect to the DNA sequence within the target editing site; (d) comprising an RNA-guided nuclease, wherein the target editing site comprises a sequence that is complementary to the PAM sequence and the guide RNA and is located immediately adjacent to the PAM sequence; (e) comprising at least one sequence-specific endonuclease, wherein the sequence-specific endonuclease provides a 5' overhang at the target edit site after cleavage. System.
  9. A system according to claim 7, wherein the genome editing molecule and HDR promoter provide modification of a target editing site of a eukaryotic cell genome using a donor template polynucleotide by HDR at a frequency at least twice as increased compared to the control group.
  10. In Clause 7, the above SSAP (a) Provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules; (b) containing RecT/Redβ-, ERF-, or RAD52- and proteins, wherein (i) The above RecT/Redβ- and protein comprises the Rac bacterial prophage RecT protein, bacteriophage λ beta protein, bacteriophage SPP1 35 protein, or an associated protein having equivalent SSAP activity; (ii) The above RecT/Redβ- and protein comprises bacteriophage λ-beta protein, bacteriophage SPP1 35 protein, Rac bacterial prophage RecT protein, or an associated protein having equivalent SSAP activity; (iii) The above RecT/Redβ- and protein comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with respect to SEQ ID NO: 1, 2 or 3; (iv) The above ERF- and protein comprises a bacteriophage P22 ERF protein, a functionally related protein, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 4; (v) The above RAD52- and protein comprises Saccharomyces cerevisiae Rad52 protein, Schizochoromyces pombe Rad22 protein, Cluyveromyces lactis Rad52 protein, a functionally related protein, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 5, 6, or 7. System.
  11. In paragraph 1 or 7, the exonuclease (a) having a preferred substrate comprising a linear dsDNA molecule; (b) having a preferred substrate comprising a linear dsDNA molecule containing a phosphorylated 5'end; (c) having 5' to 3' exonuclease activity and being able to recognize blunt-terminal dsDNA substrates, dsDNA substrates having internal breaks on one strand, dsDNA substrates having a 5' overhang and/or dsDNA substrates having a 3'overhang; (d) having 3' to 5' exonuclease activity and being able to recognize blunt-terminated dsDNA substrates, dsDNA substrates having internal breaks on one strand, dsDNA substrates having a 5' overhang and/or dsDNA substrates having a 3'overhang; (e) At least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to bacteriophage lambda exo protein, Rac prophage RecE exonuclease, Artemis protein, Apollo protein, DNA2 exonuclease, Exo1 exonuclease, herpesvirus SOX protein, UL12 exonuclease, enterobacter exonuclease VIII, T7 phage exonuclease, Escherichia coli exonuclease III, mammalian Trex2 exonuclease, related proteins having equivalent exonuclease activity or SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 containing proteins Method or system.
  12. A system according to claim 8, wherein the exonuclease comprises T7 phage exonuclease, Escherichia coli exonuclease III, an associated protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 143 or 144.
  13. In claim 1 or 7, the single-stranded DNA binding protein (SSB) (a) obtained from the same host organism as the above SSAP and/or; (b) Bacteria SSB and/or; (c) Enterobacteriaceae species SSB and/or; (d) Escherichia species, Shigella species, Enterobacter species, Klebsiella species, Serratia species, Pantoea species or Yersinia species SSB and/or; (e) a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO:31, or 34 to 132 Method or system.
  14. In paragraph 1 or 7, (a) The frequency of the HDR is increased by at least twofold compared to a control system in which genome editing molecules are provided to control eukaryotic cells but are not exposed to at least one of the HDR promoters; and/or (b) The frequency of non-homologous end-linkages (NHEJs) is maintained or at least reduced by a factor of 2 compared to a control method or system in which genome-editing molecules are provided to control eukaryotic cells but they are not exposed to at least one of the HDR promoters. Method or system.
  15. In claim 1 or 7, the SSAP, the exonuclease and/or the single-stranded DNA binding protein (SSB) (a) additionally comprising operablely coupled nuclear localization signal (NLS) and/or cell-permeable peptide (CPP); (b) provided to a cell as a polyprotein comprising a protease recognition site or a self-processed protein sequence inserted between the SSAP, the exonuclease and/or the SSB. Method or system.
  16. A method or system according to any one of claims 1 to 10 and 12, wherein the eukaryotic cell is a mammalian cell or a plant cell.
  17. In paragraph 16, the above cell is a plant cell, and here (a) The above plant cell is haploid, diploid, or polyploid; (b) the above plant cells are present in a culture medium, within a plant, or within a plant tissue; (c) The SSAP, exonuclease and/or single-stranded DNA binding protein further comprises an operablely coupled nuclear localization signal (NLS) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; (d) The system provides for isolating and/or growing a plant cell, propagule, or plant obtained from a plant cell containing a genome modification, wherein the genome of the plant cell, propagule, or plant contains a genome modification. Method or system.
  18. A method or system according to any one of claims 1 to 10 and 12, wherein an HDR promoter, a genome-editing molecule, and a eukaryotic cell or a eukaryotic cell comprising a genome modification are provided in an array comprising a plurality of containers, compartments, or locations, and each container, compartment, or location comprises an HDR promoter, a genome-editing molecule, and a eukaryotic cell or a eukaryotic cell comprising a genome modification.
  19. i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule homologous to a target editing site in a eukaryotic cell, iii) Single-stranded DNA annealing protein (SSAP), iv) an exonuclease capable of converting a double-stranded DNA substrate at least partially into a single-stranded DNA product, and v) Single-stranded DNA binding protein (SSB) A genetic engineering method for a eukaryotic cell comprising the step of providing to a eukaryotic cell, The target editing site of the above cell is modified by a donor template DNA molecule method.
  20. In paragraph 19, the above at least one sequence-specific endonuclease (a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease, wherein the method further comprises the step of providing a guide RNA or a polynucleotide encoding a guide RNA to a eukaryotic cell and/or; (b) comprising zinc-finger nucleases (ZFN), transcriptional activator-like effector nucleases (TAL-effector nucleases), argonauts, meganucleases, or engineered meganucleases; (c) Including Nikkaze method.

Description

Improved Homology-Dependent Repair Genome Editing Cross-reference regarding related applications This application claims priority to U.S. Provisional Application No. 62/866,317 filed June 25, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. Submission of a list of sequences in an ASCII text file The contents of the submission in the following ASCII text file are incorporated herein by reference in their entirety: List of sequences in computer-readable form (CRF) (filename: 165362000640SEQLIST.TXT, date recorded: June 24, 2020, size: 284 KB). Technology field The present application relates to a method, kit, and composition for gene editing. Homologous-designated repair (HDR) is a genome editing method that can be used to precisely replace target genomic DNA regions with sequences derived from provided DNA templates containing the desired replacement sequence. While the results of HDR can be quite desirable, it does not work well for a number of reasons. One of the biggest problems is its low overall occurrence rate, particularly when compared to alternative non-homologous end-linkage (NHEJ) repair mechanisms, which are often triggered by genome editing molecules that cleave targeted edit sites within the genome. Although most cells may possess several pathways capable of mediating HDR, some of these are most active during the cell cycle, which reduces the success rate of HDR under typical cell culture conditions. In prokaryotic hosts, such as Escherichia coli ( E. coli ), homologous gene replacement can be performed using a bacteriophage λ Red homologous recombination system comprising bacteriophage λ exonuclease, bacteriophage λ beta protein, a single-stranded DNA annealing protein (SSAP) that facilitates the annealing of complementary DNA strands, and a DNA template (Murphy, 2016). Recombination at target sequences within bacterial genomes has been performed by combining the bacteriophage λ Red homologous recombination system with the CRISPR-Cas9 system in prokaryotes (Jiang et al. , 2013; Wang et al. , 2016). Methods, systems, eukaryotic cells (e.g., plant cells or mammalian cells), and compositions (e.g., cell culture compositions, nucleic acids, vectors, kits, or cells) capable of providing an increased frequency of modification of a target edit site in a eukaryotic cell genome using a donor template polynucleotide by homology-designated repair (HDR) compared to a control group are disclosed herein. Features of these methods, systems, eukaryotic cells (e.g., plant cells or mammalian cells), and compositions (e.g., cell culture compositions, nucleic acids, vectors, kits, or cells) capable of providing such increased frequency of HDR include providing a single-stranded DNA annealing protein (SSAP), an exonuclease capable of converting a double-stranded DNA substrate at least partially into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB), in combination with a genome editing molecule comprising at least one sequence-specific endonuclease that cleaves a target edit site within the eukaryotic cell genome and a donor template DNA molecule having homology to the target edit site. In a specific embodiment, a donor template DNA molecule is adjacent to a copy of an endonuclease recognition sequence. The method provided herein comprises a method for increasing homology-designated repair (HDR)-mediated genomic modification of a target edit site of a eukaryotic cell genome, comprising: providing a genome-editing molecule and an HDR promoter to a eukaryotic cell; the genome-editing molecule and the HDR promoter provide modification of a target edit site of a eukaryotic cell genome using a donor template polynucleotide by HDR at an increased frequency compared to a control, wherein the genome-editing molecule comprises (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding a sequence-specific endonuclease that cleaves a DNA sequence within the target edit site; and (ii) a donor template DNA molecule having homology to the target edit site; and the HDR promoter comprises a single-stranded DNA annealing protein (SSAP), an exonuclease capable of converting a double-stranded DNA substrate at least partially into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB). The method provided herein also comprises a method for producing a eukaryotic cell having a genome modification, comprising: providing a genome editing molecule and a homology-designated repair (HDR) promoter to a eukaryotic cell; the genome editing molecule and the HDR promoter provide modification of a target editing site of the eukaryotic cell genome using a donor template polynucleotide by HDR at an increased frequency compared to a control, wherein the genome editing molecule comprises (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding a sequence-specific endonuclease that cleave