# US 2021/0277422 A1 · Homology dependent repair genome editing

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- Number: US 2021/0277422 A1
- Document ID: US20210277422A1
- Status: Published application
- Kind: A1
- Application number: 17/326,891
- Filed: 2021-05-21
- Published: 2021-09-09
- Earliest claimed: 2019-06-25
- Assignees: Inari Agriculture, Inc.
- Applicants: Inari Agriculture, Inc.
- Inventors: Tomás CERMÁK
- Web page: https://patentgrep.com/patent/US20210277422A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/902: {using homologous recombination}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim at position 1

1-173. (canceled)

### Claim 174

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).

### Claim 175 (depends on 174)

The system of claim 174, wherein the genome editing molecules or sequence-specific endonuclease is selected from the group consisting of an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and a guide RNA or a polynucleotide encoding a guide RNA.

### Claim 176 (depends on 175)

The system of claim 175, wherein the RNA-guided nuclease is selected form the group consisting of a type II Cas nuclease, a Cas9 nuclease, a type V Cas nuclease, a Cas12a nuclease, a Cas12b nuclease, a Cas12c nuclease, a CasY nuclease, a CasX nuclease, Cas12i, Cas14 and an engineered nuclease.

### Claim 177 (depends on 174)

The system of claim 174, wherein the SSAP is selected form the group consisting of a RecT/Redβ-, ERF-, and a RAD52-family protein.

### Claim 178 (depends on 177)

The system of claim 177, wherein the RecT/Redβ-family protein is selected from the group consisting of a Rac bacterial prophage RecT protein, a bacteriophage λ beta protein, and a bacteriophage SPP1 35 protein.

### Claim 179 (depends on 177)

The system of claim 177, wherein the exonuclease has 5′ to 3′ exonuclease activity and can recognize a blunt ended dsDNA substrate, a dsDNA substrate having an internal break in one strand, a dsDNA substrate having a 5′ overhang, or a dsDNA substrate having a 3′ overhang.

### Claim 180 (depends on 174)

The system of claim 174, wherein the exonuclease is selected from the group consisting of bacteriophage lambda exo protein, an Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpesvirus SOX protein, UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, E. coli Exonuclease III, a mammalian Trex2 exonuclease, and a protein having at least 70% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145.

### Claim 181 (depends on 174)

The system of claim 174, wherein the SSB has at least 70% sequence identity to SEQ ID NO: 31, 34-131, or 132.

### Claim 182 (depends on 174)

The system of claim 174, wherein the SSAP is a bacteriophage lambda beta SSAP, the exonuclease is a bacteriophage lambda exonuclease, and the SSB is an E. coli SSB.

### Claim 183 (depends on 174)

The system of claim 174, wherein the frequency of HDR is increased by at least 2-fold in comparison to a control system wherein a control plant cell is provided with the genome editing molecules but is not exposed to at least one of said HDR promoting agents.

### Claim 184 (depends on 174)

The system of claim 174, wherein the SSAP, the exonuclease, and/or the single stranded DNA binding protein further comprise an operably linked nuclear localization signal (NLS) or a cell-penetrating peptide (CPP).

### Claim 185 (depends on 184)

The system of claim 184, wherein the operably linked NLS is 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.

### Claim 186 (depends on 174)

The system of claim 174, wherein the plant cell is haploid, diploid, or polyploid.

### Claim 187 (depends on 174)

The system of claim 174, wherein the plant cell is in a culture medium, in a plant, or in a plant tissue.

### Claim 188 (depends on 174)

The system of claim 174, wherein the system provides for isolating and/or growing a plant cell, propagule, or plant obtained from the plant cell comprising the genome modification, and wherein the genome of the plant cell, propagule, or plant comprises the genome modification.

### Claim 189

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.

### Claim 190 (depends on 189)

The method of claim 189, wherein the sequence-specific endonuclease comprises an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and a guide RNA or a polynucleotide encoding a guide RNA.

### Claim 191 (depends on 190)

The method of claim 190, wherein the RNA-guided nuclease is selected from the group consisting of a type II Cas nuclease, a Cas9 nuclease, a type V Cas nuclease, a Cas12a nuclease, a Cas12b nuclease, a Cas12c nuclease, a CasY nuclease, a CasX nuclease, and an engineered nuclease

### Claim 192 (depends on 189)

The method of claim 189, wherein the donor DNA molecule is provided on a circular DNA vector, geminivirus replicon, or as a linear DNA fragment.

### Claim 193 (depends on 189)

The method of claim 189, wherein the donor DNA molecule is flanked by an endonuclease recognition sequence.

### Claim 194 (depends on 189)

The method of claim 189, wherein the SSAP is selected from the group consisting of an RecT/Redβ-, ERF-, and a RAD52-family protein.

### Claim 195 (depends on 194)

The method of claim 194, wherein the RecT/Redβ-family protein is selected from the group consisting of a Rac bacterial prophage RecT protein, a bacteriophage λ beta protein, a bacteriophage SPP1 35 protein, and a protein having at least 70% sequence identity to SEQ ID NO: 1, 2, or 3.

### Claim 196 (depends on 189)

The method of claim 189, wherein the exonuclease has 5′ to 3′ exonuclease activity and can recognize a blunt ended dsDNA substrate, a dsDNA substrate having an internal break in one strand, a dsDNA substrate having a 5′ overhang, or a dsDNA substrate having a 3′ overhang.

### Claim 197 (depends on 189)

The method of claim 189, wherein the exonuclease is selected from the group consisting of a bacteriophage lambda exo protein, an Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpesvirus SOX protein, UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, E. coli Exonuclease III, a mammalian Trex2 exonuclease, or a protein having at least 70% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145.

### Claim 198 (depends on 189)

The method of claim 189, wherein the SSB has at least 70% sequence identity to SEQ ID NO: 31, 34-131, or 132.

### Claim 199 (depends on 189)

The method of claim 189, wherein the SSAP is a bacteriophage lambda beta SSAP, the exonuclease is a bacteriophage lambda exonuclease, and the SSB is an E. coli SSB.

### Claim 200 (depends on 189)

The method of claim 189, wherein the frequency of HDR is increased by at least 2-fold in comparison to a control method wherein a control eukaryotic cell is provided with the genome editing molecules but is not exposed to at least one of said HDR promoting agents.

### Claim 201 (depends on 189)

The method of claim 189, wherein the frequency of non-homologous end-joining (NHEJ) is maintained or decreased by at least 2-fold in comparison to a control method wherein a control eukaryotic cell is provided with the genome editing molecules but is not exposed to at least one of said HDR promoting agents.

### Claim 202 (depends on 189)

The method of claim 189, wherein the eukaryotic cell is a mammalian cell or a plant cell.

### Claim 203

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.

### Claim 204 (depends on 203)

The method of claim 203, wherein the modification in selected from the group consisting of an insertion of one or more nucleotides, a deletion of one or more nucleotides, or a substitution of one or more nucleotides.

### Claim 205 (depends on 203)

The method of claim 203, wherein a portion of the target editing site is deleted by using two sequence specific cleavages in said target editing site, and is replaced by a sequence provide by the donor molecule.

### Claim 206 (depends on 203)

The method of claim 203, wherein said donor sequence is in a vector flanked by endonuclease recognition sequences.

### Claim 207 (depends on 203)

The method of claim 203, wherein the method further comprises propagating the eukaryotic cell comprising the modification.

### Claim 208 (depends on 203)

The method of claim 203, wherein the at least one SSAP is a bacteriophage lambda beta SSAP, the at least one exonuclease is a bacteriophage lambda exonuclease, and the at least one SSB is an E. coli SSB.

### Claim 209 (depends on 203)

A method of producing a genetically modified organism comprising the steps of
(i) producing a genetically modified eukaryotic cell by the method of claim 203, and
(ii) regenerating said cell into an organism.

### Claim 210 (depends on 209)

The method of claim 209, wherein the organism is selected from the group consisting of plants and non-human animals.