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US 2021/0277422 A1Published application
Homology dependent repair genome editing
AssigneeInari Agriculture, Inc.
InventorTomás CERMÁK
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Abstract
Eukaryotic cells and related reagents, systems, methods, and compositions for increasing the frequency of homology directed repair (HDR) of target editing sites with genome editing molecules are provided.
Claims38
1-173. (canceled)
A system for increasing Homology Directed Repair (HDR)-mediated genome modification of a target editing site of a plant cell, comprising:
- (a) a plant cell;
- (b) heterologous HDR promoting agents comprising a single-stranded DNA annealing protein (SSAP), an exonuclease which can convert a double stranded DNA substrate to 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 which cleaves a DNA sequence in the target editing site or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site;
- wherein the plant cell is associated with, contacts, or contains an effective amount of the HDR promoting agents and the genome editing molecule(s).
2 more elements
A method for making a eukaryotic cell having a genomic modification, comprising:
- (a) providing genome editing molecules and heterologous Homology Directed Repair (HDR) promoting agents to a eukaryotic cell, wherein the genome editing molecules comprise:
- at least one sequence-specific endonuclease which cleaves a DNA sequence in the target editing site or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site;
- and wherein the heterologous HDR promoting agents comprise a single-stranded DNA annealing protein (SSAP), an exonuclease which can convert a double stranded DNA substrate to a single stranded DNA product, and a single stranded DNA binding protein (SSB);
- whereby the genome editing molecules and HDR promoting agents provide for modification of the target editing site of the eukaryotic cell genome with the donor template DNA by HDR at a frequency that is increased in comparison to a control;
- and
- (b) isolating or propagating a eukaryotic cell comprising the genome modification, thereby making the eukaryotic cell having a genomic modification.
3 more elements
A method for producing a eukaryotic cell with a genetically modified target editing site comprising:
- (a) providing at least one sequence-specific endonuclease which cleaves a DNA sequence at least one endonuclease recognition sequence in said target editing site or at least one polynucleotide encoding said at least one sequence-specific endonuclease, and
- (b) providing at least one donor molecule comprising at least one double-stranded DNA sequence, wherein (i) said DNA sequence has a homology of at least 90% over a length of at least 50 nucleotides to sequences flanking the target editing site and (ii) wherein said donor sequence comprises at least one modification in comparison to said target editing site;
- and
- (c) providing at least one heterologous Homology Directed Repair (HDR) promoting agent comprising
- (i) at least one single-stranded DNA annealing protein (SSAP), and
- (ii) at least one exonuclease which can convert a double stranded DNA substrate to a single stranded DNA product, and
- (iii) at least one single stranded DNA binding protein (SSB);
- and whereby the at least one sequence-specific endonucleases, the at least one donor molecule, and the at least one HDR promoting agent introduce said modification into said target editing site of said eukaryotic cell;
- and
- (d) isolating a eukaryotic cell comprising a modification in said target editing site.
7 more elements
Dependencies are inferred from the claim text.