Improved homologous dependent repair genome editing

Abstract

The present invention provides improved eukaryotic cells and related reagents, systems, methods, and compositions for increasing the frequency of homologous directed repair (HDR) by genomic editing molecules to a target editing site.

Description

This application is a divisional application of the invention application with the application date of June 24, 2020, Chinese application number 202080044833.1, and invention name “Improved homology-dependent repair genome editing”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 866,317, filed on June 25, 2019, which is hereby incorporated by reference in its entirety for all purposes. Submit sequence listing as ASCII text file

[0002] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of a Sequence Listing (File Name: 165362000640SEQLIST.TXT, Record Date: June 24, 2020, Size: 284KB). Technical Field

[0003] This application relates to methods, kits, and compositions for gene editing. Background Art

[0004] Homology-directed repair (HDR) is a genome editing method that can be used to precisely replace a targeted genomic DNA site with a sequence derived from a supplied DNA template containing the desired replacement sequence. While the results of HDR are highly desirable, its effectiveness is less than ideal for a number of reasons. One of the biggest issues is its overall low frequency of occurrence, especially compared to the alternative non-homologous end joining (NHEJ) repair mechanism that is typically triggered by genome editing molecules that cut the targeted editing site in the genome. While most cells likely have multiple pathways that can mediate HDR, some of them are most active during the cell cycle, reducing the success rate of HDR under typical cell culture conditions.

[0005] In prokaryotic hosts such as Escherichia coli, homologous gene replacement can be achieved through the bacteriophage λ Red homologous recombination system, which contains bacteriophage λ exonuclease, bacteriophage λβ protein, and single-stranded DNA annealing protein (SSAP) (which facilitates the annealing of complementary DNA strands and DNA templates) (Murphy, 2016). The bacteriophage λ Red homologous recombination system has been combined with the CRISPR-Cas9 system in prokaryotes to achieve recombination of target sequences in bacterial genomes (Jiang et al., 2013; Wang et al., 2016). Summary of the Invention

[0006] Disclosed herein are methods, systems, eukaryotic cells (e.g., plant cells or mammalian cells), and compositions (e.g., cell culture compositions, nucleic acids, vectors, kits, or cells) that can provide an increased frequency of modification of a target editing site of a eukaryotic cell genome by a donor template polynucleotide via homology-directed repair (HDR) compared to a control. Features of such methods, systems, eukaryotic cells (e.g., plant cells or mammalian cells), and compositions (e.g., cell culture compositions, nucleic acids, vectors, kits, or cells) that can provide such increased HDR frequencies include providing an HDR facilitator (which comprises a single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB)) in combination with a genome editing molecule (which comprises at least one sequence-specific endonuclease that cuts a target editing site in a eukaryotic cell genome and a donor template DNA molecule having homology to the target editing site). In certain embodiments, the donor template DNA molecule is flanked by a copy of an endonuclease recognition sequence.

[0007] The methods provided herein include methods for increasing homology-directed repair (HDR)-mediated genome modification of a target editing site of a eukaryotic cell genome, the methods comprising: providing genome editing molecules and HDR promoters to a eukaryotic cell, wherein the genome editing molecules comprise: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site; and wherein the HDR promoters comprise a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); thereby the genome editing molecules and the HDR promoters provide modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide through HDR at an increased frequency compared to a control.

[0008] The methods provided herein also include methods for preparing eukaryotic cells having genome modifications, the methods comprising: providing genome editing molecules and homology-directed repair (HDR) facilitators to eukaryotic cells, wherein the genome editing molecules comprise: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; and wherein the HDR facilitators comprise a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); whereby the genome editing molecules and the HDR facilitators provide modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide at an increased frequency compared to a control through HDR; and isolating or propagating eukaryotic cells comprising the genome modification.

[0009] The systems provided herein include systems for increasing homology-directed repair (HDR)-mediated genome modification of target editing sites in the genome of a eukaryotic cell, comprising: (a) Eukaryotic cells; (b) an HDR promoter comprising a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); and (c) one or more genome editing molecules comprising at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; wherein the eukaryotic cell is associated with, contacted with and / or contains an effective amount of the HDR accelerators and the one or more genome editing molecules.

[0010] The methods provided herein also include methods for genetic engineering of eukaryotic cells, comprising providing to the eukaryotic cell: i) at least one sequence-specific nuclease, ii) a donor template DNA molecule having homology to a target editing site in the eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB), wherein the target editing site of the cell is modified by the donor template DNA molecule.

[0011] The methods provided herein also include methods for producing a eukaryotic cell having a genetically modified target editing site, comprising: (a) providing at least one sequence-specific endonuclease or at least one polynucleotide encoding the at least one sequence-specific endonuclease, which at least one sequence-specific endonuclease cleaves a DNA sequence of at least one endonuclease recognition sequence in the target editing site, and (b) providing at least one donor molecule comprising at least one double-stranded DNA sequence, wherein (i) the DNA sequence has at least 90% homology to a sequence flanking the target editing site over a length of at least 50 nucleotides and (ii) the sequence is at least 90% homologous to a sequence flanking the target editing site over a length of at least 50 nucleotides and (iii) the sequence is at least 90% homologous to a sequence flanking the target editing site. (c) providing at least one homology-directed repair (HDR) facilitator comprising (i) at least one single-stranded DNA annealing protein (SSAP), and (ii) at least one exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and (iii) at least one single-stranded DNA binding protein (SSB); and whereby the at least one sequence-specific endonuclease, the at least one donor molecule, and the at least one HDR facilitator introduce the modification into the target editing site of the eukaryotic cell; and (d) isolating the eukaryotic cell comprising the modification at the target editing site.

[0012] The compositions provided herein include compositions comprising nucleic acids encoding one or more of: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

[0013] The vectors provided herein include vectors comprising a nucleic acid encoding one or more of: i) at least one sequence-specific nuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

[0014] The kits provided herein include a kit comprising a nucleic acid encoding: i) at least one sequence-specific nuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB) and instructions for use in genetically engineering eukaryotic cells.

[0015] The cells provided herein include cells comprising i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

[0016] The cells provided herein also include a progenitor eukaryotic cell or progenitor organism for genetic engineering at a target editing site, the progenitor eukaryotic cell or progenitor organism comprising a subset of: i) at least one sequence-specific endonuclease, ii) a donor template molecule having homology to the target editing site in the eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that is capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB), wherein the cell does not comprise at least one of i)-v), wherein providing the cell or organism with the at least one of i)-v) not comprised in the progenitor cell or progenitor organism results in modification of the target editing site by the donor template molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic diagram of the vector pRS08t is shown, with base pair lengths indicated by labels on the outside of the vector. Starting from base pair 1, the vector includes a high copy number replication origin (high copy origin), a Cas expression cassette (tomato SlUBI10 promoter, Cas nuclease coding sequence (Cas nuclease CDS), and HSP terminator), a guide RNA expression cassette (Arabidopsis thaliana U6 promoter (AtU6), sequence encoding the guide RNA, and a 35S promoter), an mGFP6 sequence, a pea rbcS E9 terminator, an ANT1 donor template, and a spectinomycin resistance marker (SpnR).

[0018] Figure 2A schematic diagram of the vector pRS045 is shown. The length of the base pairs is indicated by the label outside the vector. Starting from base pair 1, the vector includes an ampicillin resistance marker (AmpR), an HDR promoter expression cassette (PcUbi promoter, c2 nuclear localization sequence (NLS) fused to the E. coli SSB coding sequence (E. coli SSB CDS), pea 3A terminator, tomato SlUBI10 promoter, c2 NLS fused to the SSAP coding sequence (RedβCDS), HSP terminator, 2x 35S promoter, c2 NLS fused to the exonuclease coding sequence (Red Exo CDS) and 35S terminator) and a pUC origin of replication (pUC ori).

[0019] Figure 3 A schematic diagram of the vector pAP046 is shown. The length in base pairs is indicated by a label on the outside of the vector. Starting from base pair 1, the vector includes a high copy number replication origin (high copy origin), a Cas expression cassette (tomato SlUBI10 promoter, a Cas nuclease coding sequence (Cas nuclease CDS), and an HSP terminator), a guide RNA and ribozyme expression cassette (35S promoter, a sequence encoding a hammerhead (HH) ribozyme, a sequence encoding a guide RNA, a sequence encoding a hepatitis delta virus (HDV) ribozyme, and a 35S terminator), an HDR promoter expression cassette (PcUbi promoter, a c2 NLS fused to an Escherichia coli SSB coding sequence (E. coli SSB CDS), a pea 3A terminator, a tomato SlUBI10 promoter, a c2 NLS fused to an SSAP coding sequence (Redβ CDS), an HSP terminator, a 2x35S promoter, a c2 NLS fused to an exonuclease coding sequence (Red Exo CDS), and a 35S terminator), an ANT1 donor template, and a spectinomycin resistance marker (SpnR).

[0020] Figure 4 Shown is a schematic diagram of vector pRS148. The length of the base pair is indicated by a tag outside the vector. Starting from base pair 1, the vector includes a high copy number replication origin (high copy origin), a Cas expression cassette (tomato SlUBI10 promoter, a Cas nuclease coding sequence (Cas nuclease CDS) and an HSP terminator), a guide RNA and a ribozyme expression cassette (a 35S promoter, a sequence encoding a hammerhead (HH) ribozyme, a sequence encoding a guide RNA, a sequence encoding a hepatitis delta virus (HDV) ribozyme and a 35S terminator) and a spectinomycin resistance marker (SpnR).

[0021] Figure 5A schematic diagram of the vector pRS192 is shown. The base pair length is indicated by a label on the outside of the vector. Starting from base pair 1, the vector includes a high copy number replication origin (high copy origin), an HDR promoter expression cassette (PcUbi promoter, c2NLS fused to the E. coli SSB coding sequence (E. coli SSB CDS), pea 3A terminator, tomato SlUBI10 promoter, c2 NLS fused to the SSAP coding sequence (RedβCDS), HSP terminator, 2x 35S promoter, c2 NLS fused to the exonuclease coding sequence (Red Exo CDS) and 35S terminator), ANT1 donor template, and an ampicillin resistance marker (AmpR).

[0022] Figure 6 A schematic diagram of the vector pTC801 is shown. The length in base pairs is indicated by a label on the outside of the vector. Starting from base pair 1, the vector includes a high copy number replication origin (high copy origin), a Cas expression cassette (maize ubiquitin (ZmUbi) promoter, a Cas nuclease coding sequence (Cas nuclease CDS), and an HSP terminator), a guide RNA and ribozyme expression cassette (35S promoter, a sequence encoding the hammerhead (HH) ribozyme, sequences encoding guide RNAs 1 and 2, a sequence encoding the hepatitis delta virus (HDV) ribozyme, and a 35S terminator), an HDR promoter expression cassette (rice actin (OsActin) promoter, a c2 NLS fused to an Escherichia coli SSB coding sequence (E. coli SSB CDS), a pea 3A terminator, a switchgrass ubiquitin (PvUbi1) promoter, a c2 NLS fused to an SSAP coding sequence (RedβCDS), a pea rbcS E9 terminator, a rice ubiquitin (OsUB1) promoter, a c2 NLS fused to an exonuclease coding sequence (Red Exo CDS) and tobacco elongation protein (NtEXT) terminators), SPX donor template, and spectinomycin resistance marker (SpnR).

[0023] Figure 7A schematic diagram of the vector pAB156 is shown. The length in base pairs is indicated by a label on the outside of the vector. Starting from base pair 1, the vector includes a kanamycin resistance marker (KanR), a left T-DNA border, a hygromycin resistance cassette (2x 35S promoter, hygromycin phosphotransferase (hygR) coding sequence and a 35S terminator), a Cas expression cassette (tomato SlUBI10 promoter, Cas nuclease coding sequence (Cas nuclease CDS) and HSP terminator), a guide RNA and ribozyme expression cassette (35S promoter, sequence encoding guide RNA, sequence encoding hammerhead (HH) ribozyme, sequence encoding hepatitis delta virus (HDV) ribozyme and a 35S terminator), an HDR promoter expression cassette (PcUbi4 promoter, c2 NLS fused to Escherichia coli SSB coding sequence (E. coli SSBCDS), pea 3A terminator, AtUbi10 promoter, c2 NLS fused to SSAP coding sequence (Redβ CDS), pea rbcS E9 terminator, HaUbiCh4 promoter, c2 NLS fused to exonuclease coding sequence (Red Exo CDS), and Ext3' terminator), GFP donor template, right T-DNA border, and STA region from pVS1.

[0024] Figure 8 Schematic diagrams of the designed insert regions for the super binary T-DNA vectors pIN1757 (bottom) and pIN1576 (top) are shown. pIN1757 includes the left T-DNA border, NOS terminator, PAT for glufosinate selection, 35S promoter, Cas expression cassette (maize ubiquitin (ZmUbi) promoter, Cas nuclease coding sequence (Cas nuclease CDS), and HSP terminator), guide RNA expression cassette (wheat U6 (TaU6) promoter, guide RNA encoding sequence (Gln1-3 Pro-2), and Pol III terminator), Gln1-3 donor template, and right T-DNA border. In addition, the vector pIN1756 includes an HDR promoter expression cassette (rice actin (OsActin promoter + intron) promoter, Escherichia coli SSB coding sequence (SSB), pea 3A terminator; switchgrass ubiquitin (PvUbi1 promoter + intron) promoter, SSAP coding sequence (β), pea rbcS E9 terminator; rice ubiquitin (OsUB1) promoter, exonuclease coding sequence (Exo) and tobacco elongation protein (NtEXT) terminator).

[0025] Figure 9A-9B A schematic diagram of the vector and expression cassette used for transformation of tomato cotyledons is shown. Figure 9AA schematic diagram of the vector pIN1705 is shown. The length in base pairs is indicated by a label on the outside of the vector. Starting from base pair 1, the vector includes a kanamycin resistance marker (KanR), a left T-DNA border, a 5-enolpyruvylshikimate-3-phosphate (EPSPS) synthase expression cassette (i.e., EPSPS coding sequence (CDS) controlled by the Arabidopsis thaliana ubiquitin promoter (AtUbi10) and the pea rbcS E9 terminator), a Cas expression cassette (tomato SlUBI10 promoter, Cas nuclease coding sequence (Cas nuclease CDS) and HSP terminator), a guide RNA and ribozyme expression cassette (35S promoter, sequence encoding hammerhead (HH) ribozyme, sequence encoding guide RNA, sequence encoding hepatitis delta virus (HDV) ribozyme, 35S terminator), an HDR promoter expression cassette (PcUbi promoter, c2 NLS fused to the Escherichia coli SSB coding sequence (E. coli SSB CDS), the pea 3A terminator, the tomato SlUBI10 promoter, c2 NLS fused to SSAP coding sequence (RedβCDS), HSP terminator, 2x 35S promoter, c2 NLS fused to exonuclease coding sequence (Red Exo CDS) and 35S terminator), ANT1 donor template, right T-DNA border, STA region from pVS1, pVS1 replication origin (ori), and replication origin (ori). Figure 9B A schematic diagram shows the region between the left and right borders of the Agrobacterium T-DNA vector used for chromosomal integration into the tomato cotyledon genome. Regions of the pIN1703, pIN1704, and pIN1705 vectors are shown from top to bottom. CS denotes the cleavage site, EPSPS denotes the EPSPS expression cassette, CasS denotes the Cas expression cassette, ANT1 Donor denotes the donor template, HDR Agent denotes the HDR promoter expression cassette encoding SSAP, SSB, and exonuclease, and GFP denotes the green fluorescent protein coding sequence.

[0026] Figure 10Shown is the schematic diagram of the carrier for expressing in people.The length of base pair is indicated by the label outside carrier.Starting from base pair 1, this carrier includes high copy number replication origin (high copy origin), Cas expression cassette (CAG promoter, Cas nuclease coding sequence (Cas nuclease CDS) and rabbit β-globin (rb globin) terminator), guide RNA expression cassette (Homo sapiens U6 (HsU6) promoter, the sequence of coding guide RNA), HDR promoter expression cassette (Homo sapiens EF1a promoter, SV40 NLS are connected to Escherichia coli SSB coding sequence (Escherichia coli SSB CDS), human growth hormone (hGH) terminator, Homo sapiens ACTB (hACTB) promoter, SV40 NLS are connected to SSAP coding sequence (Red β CDS), bovine growth hormone (bGH) terminator, CMV promoter, SV40 NLS are connected to exonuclease coding sequence (Red Exo CDS) and SV40 poly A signal), EMX1 FRT donor template and spectinomycin resistance marker (SpnR). DETAILED DESCRIPTION I. Definition

[0027] Unless otherwise indicated, nucleic acid sequences in the text of this specification are presented in a 5' to 3' orientation when reading from left to right. Nucleic acid sequences can be provided in the form of DNA or RNA, as specifically described; as known to those of ordinary skill in the art, disclosure of one necessarily defines the other, and necessarily defines the precise supplement. Where a term is provided in the singular, the inventors also contemplate embodiments described by the plural form of that term.

[0028] As used herein, the phrase "allelic variant" refers to polynucleotide or polypeptide sequence variations that occur in different strains, varieties, or isolates of a given organism.

[0029] Furthermore, as used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without each other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] As used herein, the terms "Cpf1" and "Cas12a" are used interchangeably herein to refer to the same RNA-directed nuclease.

[0031] As used herein, the phrase "genome editing molecule" refers to one or more sequence-specific endonucleases or one or more polynucleotides encoding the one or more sequence-specific endonucleases, which cleave at least one DNA sequence at an endonuclease recognition site.

[0032] As used herein, an "exogenous" agent or molecule refers to any agent or molecule from an external source that is provided or introduced into a system, composition, eukaryotic or plant cell culture, reaction system, or eukaryotic or plant cell. In certain embodiments, an exogenous agent (e.g., a polynucleotide, protein, or compound) from an external source can be an agent that is also present in a eukaryotic or plant cell. In certain embodiments, an exogenous agent (e.g., a polynucleotide, protein, or compound) from an external source can be an agent that is heterologous to a eukaryotic or plant cell.

[0033] As used herein, a "heterologous" agent or molecule refers to any agent or molecule that is: (i) not found in a wild-type, untreated, or naturally occurring composition, eukaryotic cell, or plant cell; and / or (ii) a polynucleotide or peptide sequence located, for example, in a genome or vector, in a context that is different from that in which the sequence occurs in nature. For example, a promoter operably linked to a gene different from the gene to which the promoter is naturally operably linked is a heterologous promoter.

[0034] As used herein, the terms "include," "includes," and "including" should be interpreted as implying at least the features they specify without excluding any additional unspecified features.

[0035] As used herein, the term "homologous recombination" refers to the exchange of DNA fragments at homologous sites between two DNA molecules. The frequency of homologous recombination is affected by a variety of factors. Different organisms differ in the number of homologous recombination events and the relative proportion of homologous and non-homologous recombination. Generally, the length of the homologous region affects the frequency of homologous recombination events: the longer the homologous region, the greater the frequency. The length of the homologous region required for observing homologous recombination is also species-variable. In many cases, at least 5kb of homology has been used, but homologous recombination has been observed with as little as 25-50bp of homology.

[0036] As used herein, homology-directed repair (HDR) refers to a DNA repair method that results in precise editing of the target editing site through the incorporation of a provided donor sequence.

[0037] As used herein, phrases such as "frequency of HDR", "HDR frequency", etc. refer to the number of HDR-mediated events at a target editing site compared to the total number of target editing sites analyzed. The total number of target editing sites is: (a) target editing sites with NHEJ-mediated events; (b) target editing sites with no changes; and (c) target editing sites with HDR-mediated events. HDR-mediated events include the precise insertion of a heterologous sequence into the target editing site without any unexpected nucleotide insertions, deletions, or substitutions in the inserted heterologous sequence, the homologous sequences flanking the heterologous insert, or the sequence at the junction of the heterologous and homologous sequences.

[0038] As used herein, the phrase "eukaryotic cell" refers to any cell that contains a nucleus and thus includes mammalian (e.g., human, livestock, and companion animal cells), insect cells, reptile cells, plant cells (e.g., monocotyledonous and dicotyledonous plant cells), yeast cells, and fungal cells (e.g., filamentous and non-filamentous fungi).

[0039] "Modified nucleotides" or "edited nucleotides" refer to a nucleotide sequence of interest that comprises at least one alteration when compared to its unmodified nucleotide sequence. Such "alterations" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0040] As used herein, the phrase "plant cell" can refer to a plant cell having a plant cell wall or a plant cell protoplast lacking a plant cell wall.

[0041] As used herein, the term "polynucleotide" is a nucleic acid molecule comprising two (2) or more nucleotide residues. Polynucleotides are typically described as single-stranded or double-stranded. When a polynucleotide comprises a double-stranded region formed by intramolecular or intermolecular hybridization, the length of each double-stranded region is conveniently described in terms of the number of base pairs. Embodiments of the systems, methods, and compositions provided herein may employ or include: (i) one or more polynucleotides having a length of 2 to 25 residues, one or more polynucleotides having a length of greater than 26 residues, or a mixture of the two. A polynucleotide may comprise single-stranded or double-stranded RNA, single-stranded or double-stranded DNA, a double-stranded DNA / RNA hybrid, a chemically modified analog thereof, or a mixture thereof. In certain embodiments, a polynucleotide may comprise a combination of ribonucleotides and deoxyribonucleotides (e.g., a synthetic polynucleotide consisting primarily of ribonucleotides but having one or more terminal deoxyribonucleotides or a synthetic polynucleotide consisting primarily of deoxyribonucleotides but having one or more terminal dideoxyribonucleotides), or may include non-canonical nucleotides, such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotides include chemically modified nucleotides (see, e.g., Verma and Eckstein (1998) Annu. Rev. Biochem., 67:99-134). Chemically modified nucleotides that can be used in the polynucleotides provided herein include: (i) phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications of the phosphodiester backbone; (ii) nucleosides comprising modified bases and / or modified sugars; and / or (iii) detectable labels including fluorescent moieties (e.g., fluorescein or rhodamine or fluorescence resonance energy transfer or FRET chromophore label pairs) or other labels (e.g., biotin or isotopes). The polynucleotides provided or used herein also include modified nucleic acids, particularly modified RNAs, as disclosed in U.S. Pat. No. 9,464,124, incorporated herein by reference in its entirety.

[0042] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences from non-AAV sources), the one or more heterologous sequences being flanked by at least one, and in some embodiments, two, AAV inverted terminal repeats (ITRs). This rAAV vector can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or expresses suitable helper functions) and expresses AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector (e.g., a plasmid for cloning or transfection)), the rAAV vector can be referred to as a "proto-virus," which, in the presence of AAV packaging functions and suitable helper functions, can be "rescued" by replication and encapsidation. The rAAV vector can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated viral particles (rAAV particles)."

[0043] "Recombinant adenoviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-adenoviral origin), the one or more heterologous sequences being flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITR). When these recombinant viral vectors are present in host cells expressing essential adenoviral genes (e.g., E1 gene, E2 gene, E4 gene, etc.) deleted from the recombinant viral genome, they can replicate and be packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector (e.g., a plasmid for cloning or transfection)), the recombinant viral vector can be referred to as a "protovirus," which, in the presence of adenoviral packaging functions, can be "rescued" by replication and encapsidation. The recombinant viral vector can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles, such as adenoviral particles. The recombinant viral vector can be packaged into an adenoviral capsid to generate a "recombinant adenoviral particle."

[0044] A "recombinant lentiviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not derived from a lentivirus) flanked by at least one lentiviral terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral terminal repeats (LTRs). Such a recombinant viral vector, when present in a host cell infected with appropriate helper functions, can replicate and be packaged into infectious viral particles. The recombinant lentiviral vector can be packaged into a lentiviral capsid to generate a "recombinant lentiviral particle."

[0045] "Recombinant herpes simplex vector (recombinant HSV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not derived from HSV) flanked by HSV terminal repeats. Such a recombinant viral vector can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with appropriate helper functions. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector (e.g., a plasmid for cloning or transfection)), the recombinant viral vector can be referred to as a "proto-virus," which can be "rescued" by replication and encapsidation in the presence of an HSV packaging function. The recombinant viral vector can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles, such as HSV particles. The recombinant viral vector can be packaged into an HSV capsid to generate a "recombinant herpes simplex virus particle."

[0046] As used herein, the phrase "target editing site" refers to a DNA sequence that is modified by a donor nucleic acid.

[0047] As used herein, the phrase "target gene" may refer to a gene located in a genome that is modified by the gene editing molecules provided in the systems, methods, compositions and / or eukaryotic cells provided herein. Embodiments of target genes include (protein) coding sequences, non-coding sequences, and combinations of coding and non-coding sequences. The modification of the target gene includes nucleotide substitutions, insertions, and / or deletions in one or more elements of the gene, including transcription enhancers or promoters, 5' or 3' untranslated regions, mature or precursor RNA coding sequences, introns, splicing donors and / or receptors, protein coding sequences, polyadenylation sites, and / or transcription terminators. In certain embodiments, all copies or all alleles of a given target gene in a diploid or polyploid plant cell are modified to provide homozygosity for the modified target gene in the plant cell. In embodiments where a desired trait is imparted by introducing a loss-of-function mutation in a target gene via gene editing, the plant cell, plant cell population, plant, or seed is homozygous for the modified target gene with a loss-of-function mutation. In other embodiments, only a copy of a given target gene or a subset of alleles is modified to provide the heterozygosity of the modified target gene in the plant cell. In certain embodiments of the desired proterties by introducing a dominant mutation of the target gene via gene editing, plant cells, plant cell colonies, plants or seeds are heterozygous for the modified target gene with a dominant mutation. The proterties given by such modifications of some plant target genes include improving yield, resistance to insects, fungi, bacterial pathogens and / or nematodes, herbicide tolerance, abiotic stress tolerance (for example, drought, cold, salt and / or heat tolerance), protein quantity and / or quality, starch quantity and / or quality, lipid quantity and / or quality, secondary metabolite quantity and / or quality, all of which are compared to control plants lacking the modification. Plants with genomes modified by gene editing molecules provided in systems, methods, compositions and / or plant cells provided herein are different from plants with genomes modified by traditional breeding (i.e., hybridization of male and female plants), wherein the unwanted and random exchange of genomic regions and the genetic and epigenetic changes produced by random mitosis or meiosis in the genome typically occur during hybridization and are then found in offspring plants. Therefore, in embodiments of plants (or plant cells) with modified genomes, the identity of the modified genome to the original (unmodified) genome exceeds 99.9%. In embodiments, the modified genome does not have genetic or epigenetic changes produced by random mitosis or meiosis relative to the original (unmodified) genome. In embodiments, the modified genome includes differences in epigenetic changes in a genome less than 0.01% relative to the original (unmodified) genome.In embodiments, the modified genome comprises: (a) a difference in DNA methylation in less than 0.01% of the genome relative to the original (unmodified) genome; or (b) a difference in DNA methylation in less than 0.005% of the genome relative to the original (unmodified) genome; or (c) a difference in DNA methylation in less than 0.001% of the genome relative to the original (unmodified) genome. In embodiments, the gene of interest is located on a chromosome in a plant cell, and the modified genome comprises: (a) a difference in DNA methylation in less than 0.01% of the genome contained within the chromosome comprising the gene of interest relative to the original (unmodified) genome; (b) a difference in DNA methylation in less than 0.005% of the genome contained within the chromosome comprising the gene of interest relative to the original (unmodified) genome; or (c) a difference in DNA methylation in less than 0.001% of the genome contained within the chromosome comprising the gene of interest relative to the original (unmodified) genome. In embodiments, the modified genome has no more unexpected changes than 1×10^-8 mutations / base pair / copy compared to the original (unmodified) genome. In certain embodiments, the modified genome has no more unexpected changes than would occur at the natural mutation rate. The natural mutation rate can be determined empirically or as described in the literature (Lynch, M., 2010; Clark et al., 2005).

[0048] As used herein, "vector" refers to a recombinant plasmid comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0049] To the extent that any of the foregoing definitions are inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, in any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the foregoing definitions will control. II. Methods and Compositions A. Methods for increasing homology-directed repair-mediated genome modifications

[0050] Provided herein are various reagents, systems, methods, and compositions comprising HDR promoters (SSAPs, exonucleases, and SSBs) and genome editing molecules and providing increased homology-dependent repair (HDR) frequencies in eukaryotic cell gene editing experiments compared to control experiments. In certain embodiments, the frequency of HDR is increased by at least 2 times, 3 times, 5 times, or 10 times compared to a control method (wherein the control eukaryotic cells have genome editing molecules, but are not exposed to at least one HDR promoter (SSAP, exonucleases, and SSBs)). In certain embodiments, the frequency of HDR is increased by at least 2 times, 3 times, or 5 times to about 12 times, 15 times, 20 times, 25 times, or 30 times compared to a control method (wherein the control eukaryotic cells have genome editing molecules, but are not exposed to at least one HDR promoter (SSAP, exonucleases, and SSBs). In some embodiments, the present method can be used for cells that have not undergone mitosis or meiosis. In some embodiments, the present method does not require DNA replication. i. Nuclear localization signal (NLS)

[0051] Nuclear localization signals (NLSs) that can direct the SSAPs, exonucleases, SSBs, and / or gene editing molecules provided herein include single-component and two-component nuclear localization signals (Kosugi et al., 2009). Examples of single-component NLSs that can be used include NLSs comprising at least 4 consecutive basic amino acids, such as the SV40 large T antigen NLS (PKKKRKV; SEQ ID NO: 11) and another type having only three basic amino acids (which has a K(K / R)X(K / R) consensus sequence (SEQ ID NO: 12). Examples of two-component NLSs that can be used herein include (K / R)(K / R)X 10-12 (K / R) 3 / 5 (SEQ ID NO: 13), wherein (K / R) 3 / 5 At least three of five consecutive amino acids, lysine or arginine, may be present. The NLS may also comprise a plant-specific Class 5 NLS having the consensus sequence LGKR(K / R)(W / F / Y) (SEQ ID NO: 14). Examples of specific NLSs that may further be used include the maize opaque-2 nuclear localization signal (SEQ ID NO: 10), the bhendi yellow vein mosaic virus (BYVMV) c2 NLS (SEQ ID NO: 15), and the extended SV40 large T antigen NLS (SEQ ID NO: 16).

[0052] In some embodiments, the NLS is a mammalian (e.g., human) NLS. In some embodiments, the NLS is an SV40 NLS. In some embodiments, the NLS is an SV40 NLS with an amino acid linker. In some embodiments, the NLS has the amino acid sequence MAPKKKRKVGGSGS (SEQ ID NO: 148).

[0053] In certain embodiments, NLS elements or other desired elements (e.g., epitope tags) can be operably connected to SSAP, exonuclease, SSB and / or gene editing molecules provided herein by direct covalent connection of elements and domains or by using linker peptides or flexible hinge polypeptides. Flexible hinge polypeptides include peptide sequences rich in glycine or containing glycine / serine. Such sequences may include, but are not limited to, (Gly4)n sequences, (Gly4Ser)n sequences, Ser(Gly4Ser)n sequences, combinations thereof, and variants thereof, wherein n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, such hinge peptides rich in glycine or containing glycine / serine may also include threonyl and / or alanyl residues (to improve flexibility) and polar lysyl and / or glutamyl residues. Other examples of hinge peptides that can be used include immunoglobulin hinge peptides (Vidarsson et al., 2014).

[0054] A variety of cell penetrating peptides (CPPs) can also be used for the SSAPs, exonucleases, SSBs and / or gene editing molecules provided herein. The CPPs that can be used include a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT containing YGRKKRRQRRR; SEQ ID NO: 17); a polyarginine sequence containing several arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) sufficient to directly enter the cell; a VP22 domain (Zender et al. (2002) Cancer Gene Ther [Cancer Gene Ther]. 9(6):489-96); Drosophila Antennary protein transduction domain (Noguchi et al. (2003) Diabetes [Diabetes] 52(7):1732-1737); truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research [Drug Research] 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:18); transportan (e.g., GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:19); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:20); and RQIKIWFQNRRMKWKK (SEQ ID Exemplary CPP amino acid sequences also include YGRKKRRQRRR (SEQ ID NO: 22; RKKRRQRR (SEQ ID NO: 23); YARAAARQARA (SEQ ID NO: 24); THRLPRRRRRR (SEQ ID NO: 25); and GGRRARRRRRR (SEQ ID NO: 26). ii. Single-stranded DNA annealing protein (SSAP)

[0055] In certain embodiments, the single-stranded DNA annealing proteins (SSAPs) used in the methods, systems, cells, and cell culture compositions provided herein include proteins that promote or catalyze DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules. Characteristics of the SSAPs used herein include stimulation of RecA-dependent and -independent pathways, in vitro oligomeric ring and / or filament formation, ssDNA binding activity, and non-ATPase-dependent stimulation of annealing of complementary ssDNA strands. Murphy, 2016 and Iyer et al., 2002 have disclosed characteristics of SSAP proteins in the RecT / Redβ protein family, the ERF protein family, or the RAD52 protein family. In certain embodiments, the SSAP is a member of the RecT / Redβ protein family, which includes the Rac bacterial prophage RecT protein, the phage λβ protein, the phage SPP1 35 protein, or a related protein with equivalent SSAP activity. Characteristics of certain RecT / Redβ protein families include an α+β domain with a core of five β strands and five α helices, a Mg +2 In certain embodiments, the RecT / Redβ-family protein comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, 2 or 3 and optionally a conserved α+β domain (having a core of five β strands and five α helices), Mg +2In certain embodiments, the SSAP is an ERF family protein. The EFR protein family is characterized by a conserved region of approximately 150 amino acid residues comprising the motif GuXXoYhp+YXhXXhh (SEQ ID NO: 32), wherein G is glycine, Y is tyrosine, u is a "tiny" residue (glycine, serine, alanine), h is hydrophobic (alanine, valine, leucine, isoleucine, phenylalanine, methionine), p is a polar residue (lysine, arginine, glutamic acid, aspartic acid, asparagine, threonine, serine), o is an alcohol-containing amino acid residue (serine or threonine), + is a basic residue, and X is any residue (Iyer et al., 2002). ERF family proteins include bacteriophage P22 ERF proteins or proteins having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4, and may optionally further comprise a GuXXoYhp+YXhXXhh (SEQ ID NO: 32) motif. SSAPs in the ERF family also include proteins listed in the NCBI database at the World Wide Web website ncbi.nlm.nih.gov / protein under accession numbers (gi or gene identifier) ​​9634188, 9635694, 16804357, 12719409, 458219, 11497308, 11497280, 1497168, 11527300, 9634634, 9635643, 13491642, 6015511, 11138335, 9627938, 9628668, and 15088753. In certain embodiments, the SSAPs used herein include RAD52 family proteins from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis, and variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity over the full length of SEQ ID NOs: 5, 6, and 7, respectively; or variants having one or more conservative and / or semi-conservative amino acid substitutions in SEQ ID NOs: 5, 6, or 7. The RAD52 protein family is characterized by a conserved helix-hairpin-helix (HhH) motif with DNA binding activity (Iyer et al., 2002). The SSAPs used herein may further include proteins identified as "recombinases," which are listed in at least Tables 1, 2, 3, 4, 5, and 6 of U.S. Patent Application Serial No. 16 / 075,281 (U.S. National Phase of PCT / US2017 / 016184, published as WO 2017 / 184227, incorporated herein by reference in its entirety). In certain embodiments, a SSAP may include an allelic variant of any of the above-described SSAPs.In certain embodiments, any of the aforementioned SSAPs can be provided to cells via nucleic acids encoding the SSAP (e.g., expression vectors, mRNA, or viral expression vectors). In certain embodiments, any of the aforementioned SSAPs can be provided to cells as a protein, a fusion protein (e.g., with a cell-penetrating peptide and / or a nuclear localization sequence), or as a multimeric protein comprising a protease recognition site or a self-processing protein sequence inserted between the SSAP and other proteins (e.g., in combination with SSB and / or exonucleases). iii. Exonucleases

[0056] In certain embodiments, the exonucleases used in the methods, systems, cells, and cell culture compositions provided herein include exonucleases that have 5' to 3' or 3' to 5' exonuclease activity on double-stranded DNA (dsDNA) substrates, which can produce products comprising at least a portion of single-stranded DNA (ssDNA) having an exposed 3' end or an exposed 5' end, respectively. In certain embodiments, the exonucleases will recognize dsDNA substrates with blunt ends, including blunt ends with 5' phosphate groups. In certain embodiments, the exonucleases will recognize dsDNA substrates with ssDNA overhangs (e.g., 5' or 3' ssDNA regions at the ends of dsDNA molecules, including ends produced by endonucleases that provide staggered cuts in the dsDNA substrate). In certain embodiments, the exonucleases will recognize dsDNA substrates with internal breaks in one chain (e.g., nicked dsDNA). Exonucleases with 5' to 3' exonuclease activity that can be used herein include bacteriophage lambda exo protein (e.g., SEQ ID NO: 8), Rac prophage RecE exonuclease protein (e.g., SEQ ID NO: 9), Artemis protein (e.g., SEQ ID NO: 136), Apollo protein (e.g., SEQ ID NO: 137), DNA2 exonuclease protein (e.g., SEQ ID NO: 138), Exo1 exonuclease protein (e.g., SEQ ID NO: 139), herpes virus SOX protein (e.g., SEQ ID NO: 140), UL12 exonuclease protein (e.g., SEQ ID NO: 141), enterobacterial exonuclease VIII protein (e.g., SEQ ID NO: 142), T7 phage exonuclease protein (e.g., SEQ ID NO: 143), or related proteins with equivalent 5' to 3' exonuclease activity, or proteins with the same or similar properties as SEQ ID NO: 144. NO: 8, 9, 136, 137, 138, 139, 140, 141, 142 or 143. In certain embodiments, the exonucleases provided herein having 5' to 3' exonuclease activity include proteins listed in SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142 or 143 having at least one or more conservative and / or semi-conservative amino acid substitutions in SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142 or 143.Exonucleases with 3' to 5' exonuclease activity useful herein include E. coli exonuclease III protein (e.g., SEQ ID NO: 144), mammalian Trex2 exonuclease protein (e.g., SEQ ID NO: 145), related proteins with equivalent 3' to 5' exonuclease activity, or proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 144 or 145. In certain embodiments, the exonucleases with 3' to 5' exonuclease activity provided herein include proteins listed in SEQ ID NO: 144 or 145 having at least one or more conservative and / or semi-conservative amino acid substitutions in SEQ ID NO: 144 or 145. In certain embodiments, the exonucleases will comprise the conserved DEDD catalytic residues characteristic of the DEDD / DnaQ superfamily of exonucleases (Bernad et al., 1989). In certain embodiments, any of the aforementioned exonucleases can be provided to cells as proteins, fusion proteins (e.g., with cell penetrating peptides and / or nuclear localization sequences) or as polyproteins comprising protease recognition sites or self-processing protein sequences (e.g., in combination with SSB and / or SSAP) inserted between the exonucleases and other proteins. In certain embodiments, the exonucleases can include allelic variants of any of the aforementioned exonucleases. In certain embodiments, any of the aforementioned exonucleases can be provided to cells by nucleic acids encoding the exonucleases (e.g., expression vectors, mRNA, or viral expression vectors). In certain embodiments, the sequence-specific endonuclease is a nickase. iv. Single-stranded DNA binding protein (SSB)

[0057] Various single-stranded DNA binding proteins (SSBs) can be used in the methods, systems, cells, and cell culture compositions provided herein. In certain embodiments, the SSBs include bacterial SSBs or optionally Enterobacteriaceae species SSBs. In certain embodiments, the SSBs are Escherichia sp., Shigella sp., Enterobacter sp., Klebsiella sp., Serratia sp., Pantoea sp., or Yersinia sp. The SSBs provided herein include those set forth in SEQ ID NO: 31 and SEQ ID NOs: 34-131 and 132, and variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity over the full length of SEQ ID NO: 31, SEQ ID NOs: 34-131, or 132; or variants thereof having one or more conservative and / or semi-conservative amino acid substitutions in SEQ ID NO: 31, or SEQ ID NOs: 34-131, or 132. The SSBs used herein may include the SSB proteins listed in at least Tables 7 and 8 of the present disclosure and U.S. Patent Application Serial No. 16 / 075,281 (U.S. National Phase of PCT / US2017 / 016184, published as WO 2017 / 184227, incorporated herein by reference in its entirety). In certain embodiments, the SSBs may include allelic variants of any of the above-described SSBs. In certain embodiments, any of the aforementioned SSBs can be provided to cells via nucleic acids encoding the SSBs (e.g., expression vectors, mRNA, or viral expression vectors). In certain embodiments, any of the aforementioned SSBs can be provided to cells as proteins, fusion proteins (e.g., with cell-penetrating peptides and / or nuclear localization sequences), or as polyproteins comprising a protease recognition site or a self-processing protein sequence inserted between the SSB and other proteins (e.g., in combination with SSAPs and / or exonucleases).

[0058] In some embodiments, the SSB and SSAP used in the present methods are derived from the same organism or from a bacteriophage and the bacterial host of the bacteriophage.

[0059] In some embodiments, an SSB is not required. In some embodiments, the SSAP is fused to a replication protein A (RPA) binding partner (Fanning et al., Nucleic acids research, 34(15), 4126-4137). In some embodiments, the SSB is an endogenous SSB. In some embodiments, a SSAP is provided that is modified to bind to an endogenous SSB.

[0060] In some embodiments, the components used in the methods provided herein are provided as fusion proteins. In some embodiments, SSAP is fused to SSB. In some embodiments, SSAP is fused to replication protein A (RPA). v. Plants, plant tissues and plant cells

[0061] In certain embodiments, HDR in isolated plant cells or plant protoplasts is increased (i.e., not located in unisolated or complete plant tissue, plant part, or whole plant). In certain embodiments, plant cells are obtained from any plant part or tissue or callus. In certain embodiments, the culture includes plant cells obtained from: plant tissue, cultivated plant tissue explants, whole plants, complete node buds, stem tips or stem tip meristems, root tips or root tip meristems, lateral tissues, intercalary meristems, seedlings, whole seeds, half seeds or other seed fragments, zygotic embryos, somatic embryos, immature embryos, ovules, pollen, microspores, anthers, hypocotyls, cotyledons, leaves, petioles, stems, tubers, roots, callus, or plant cell suspensions. In certain embodiments, plant cells are derived from the L1 or L2 layers of immature or mature embryos of monocotyledons (e.g., maize, wheat, sorghum, or rice).

[0062] In certain embodiments, the HDR in the plant cell in unseparated or complete plant tissue, plant part, plant explant or whole plant increases.In certain embodiments, plant cell can be positioned at following: complete node bud, cultivated plant tissue explant, stem tip or stem tip meristem, root tip or root tip meristem, lateral tissue, intercalary meristem, seedling, whole seed, half seed or other seed fragment, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root or callus.In certain embodiments, the explant used includes immature embryo.Immature embryo (for example immature corn embryo) includes 1.8-2.2mm embryo, 1-7mm embryo and 3-7mm embryo.In certain embodiments, above-mentioned embryo is obtained from the seed of mature female spike source, leaf base, leaf from mature plant, leaf tip, immature inflorescence, tassel, immature female spike and filament. In various aspects, the explant for conversion of plant origin includes immature embryo, 1.8-2.2mm embryo, 1-7mm embryo and 3.5-7mm embryo. On the one hand, the embryo for disclosed method can be derived from the seed, leaf base, leaf from mature plant, leaf tip, immature inflorescence, tassel, immature female spike and filament in mature female spike source. In certain embodiments, plant cell is pluripotent plant cell (for example, stem cell or meristematic cell). In certain embodiments, plant cell is positioned at the immature embryo of monocotyledonous plant (for example maize, wheat, sorghum or rice) or the L1 or L2 layer of mature embryo. In certain embodiments, the method disclosed in WO 2018085693 (being incorporated herein by reference in its entirety) to whole plant, seed, embryo, explant or meristematic genome being edited can be applicable to plant cell and related system, method, composition or culture provided herein.

[0063] In certain embodiments, the plant cell may comprise a haploid, diploid, or polyploid plant cell or plant protoplast, such as those obtained from a haploid, diploid, or polyploid plant, plant part or tissue, or callus tissue. In certain embodiments, the plant cells in culture (or regenerated plants, progeny seeds, and progeny plants) are haploid or can be induced to become haploid; techniques for making and using haploid plants and plant cells are known in the art, see, for example, methods for generating haploids in Arabidopsis thaliana by crossing a wild-type line with a haploid-inducing line expressing an altered form of the centromere-specific histone CENH3, as described in Maruthachalam and Chan in “How to make haploid Arabidopsis thaliana,” a protocol available at www[dot]openwetware[dot]org / images / d / d3 / Haploid_Arabidopsis_protocol[dot]pdf; (Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038 / ncomms6334). Haploids can also be obtained in a wide variety of monocots (e.g., maize, wheat, rice, sorghum, barley) or dicots (e.g., soybean, Brassica species, including canola, cotton, tomato) by crossing plants containing a mutated CENH3 gene with wild-type diploid plants to produce haploid progeny, as disclosed in U.S. Pat. No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid-induced maize lines that can be used to obtain haploid maize plants and / or cells include Stock 6, MHI (Moldovian Haploid Inducer), indeterminate gametophyte (ig) mutations, KEMS, RWK, ZEM, ZMS, KMS, and the transgenic haploid inducer lines disclosed in U.S. Pat. No. 9,677,082, which is incorporated herein by reference in its entirety. Examples of haploid cells include, but are not limited to, plant cells obtained from haploid plants and plant cells obtained from reproductive tissue, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, megagametophytes, and microspores.In certain embodiments where the plant cell or plant protoplast is haploid, the genetic complement can be doubled to produce a doubled haploid plant cell or plant protoplast, wherein the complement of the gene or allele is homozygous, by performing chromosome doubling (e.g., spontaneous chromosome doubling by meiosis without reduction, or by using a chromosome doubling agent, such as colchicine, oryzalin, trifluralin, natron, nitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in the plant cell or plant protoplast, wherein the complement of the gene or allele is homozygous; yet other embodiments include regenerating a double haploid plant from a double haploid plant cell or plant protoplast. Another embodiment relates to a hybrid plant having at least one parent plant, the parent plant being a double haploid plant provided by the method. The generation of double haploid plants provides homozygosity within one generation, without the need for several generations of self-pollination to obtain homozygous plants. In any case where it is desired to establish genetic purity (i.e., homozygosity) in the shortest possible time, it is advantageous to use double haploids. Doubled haploid production is particularly advantageous in slow-growing plants, such as fruit and other trees, or for producing hybrid plants that are the offspring of at least one doubled haploid plant.

[0064] In certain embodiments of increased HDR in plant cells, and related methods, systems, compositions, or reaction mixtures provided herein, can include plant cells obtained from or located in any monocot or dicot species of interest, such as field crop plants, fruit plants and trees, vegetables, trees, and ornamental plants, including ornamental flowers, shrubs, trees, ground cover plants, and turf grasses. In certain non-limiting embodiments, the plant material is obtained from or is located in alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cocoa (Theobroma cacao), canola and rapeseed or rapeseed (Brassicanapus), carnation (Dianthus caryophyllus), carrot (Daucus carota sativus), cassava (Manihot esculentum), cherries (Prunus avium), chickpeas (Cider arietinum), endive (Cichorium intybus), red peppers and other peppers of the genus Capsicum (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemums (Chrysanthemum spp.), coconuts (Cocos nucifera), coffee (Coffea spp.), including Coffea arabica (Coffea spp.). arabica) and Coffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), blackcurrant and gooseberry (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linumus itatissumum L.), geranium (Pelargonium spp.), grapefruit (Citrus paradisi), grapevine (Vitus spp.) (including wine grape (Vitus vinifera)), guava (Psidium guajava), hemp, hops (Humulus lupulus), iris (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.), corn (Zea mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis melo), millet (Setaria spp.), barnyard grass (Echinochloa spp.), (Eleusine spp.), Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), olives (Oleaeuropaea), onions (Allium cepa), oranges (Citrus sinensis), papayas (Carica papaya), peaches and nectarines (Prunus persica), pears (Pyrus spp.), peas (Pisa sativum), peanuts (Arachis hypogaea), peonies (Paeonia spp.), morning glories (Petunia spp.), pineapples (Ananascomosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa), poplar (Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbita pepo), rice (Oryza sativa L.), rose (Rosa spp.), rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L.), sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycine max L.), squash (Cucurbita pepo), pepo), strawberries (Fragaria spp., Fragaria xananassa), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus annus), sweet potato (Ipomoea batatas), orange (Citrustangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum), tulip (Tulipa spp.), radish (Brassica rapa rapa), walnut (Juglans spp. L.), watermelon (Citrulus lanatus), wheat (Tritium aestivum), or yam (Discorea spp.). vi. Eukaryotic cells

[0065] In certain embodiments, eukaryotic cells (e.g., plant cells) in which HDR is increased can be cells that are (a) encapsulated or enclosed in or attached to a polymer (e.g., pectin, agarose, or other polysaccharide) or other support (solid or semi-solid surface or matrix, or particles or nanoparticles); (b) encapsulated or enclosed in or attached to a vesicle or liposome or other fluid compartment; (c) not encapsulated, enclosed, or attached. In certain embodiments, the cells can be cultured in liquid or suspension culture, or in or on a semi-solid or solid culture medium, or in a combination of liquid and solid or semi-solid culture medium (e.g., plant cells or protoplasts are cultured on a solid culture medium with a liquid culture medium overlay, or plant cells or protoplasts are attached to solid beads or a matrix and grown in a liquid culture medium). In certain embodiments, cells are encapsulated in polymers (e.g., pectin, agarose, or other polysaccharides) or other encapsulating materials, encapsulated in vesicles or liposomes, suspended in a mixed phase medium (e.g., an emulsion or reverse emulsion), or embedded in or attached to a matrix or other solid support (e.g., beads or microbeads, membranes, or solid surfaces).

[0066] In a related aspect, the present disclosure provides arrangements of eukaryotic cells (e.g., plant cells) with improved HDR frequencies in the systems, methods, and compositions described herein, such as arrangements of cells for screening purposes or for high-throughput and / or multiple transformation or gene editing experiments. In one embodiment, the present disclosure provides an arrangement of multiple cells, comprising: (a) an HDR promoter; and optionally (b) a genome editing molecule. In certain embodiments, the arrangement of cells may further comprise at least one chemical, enzymatic, or physical delivery agent. In another embodiment, the present disclosure provides an array comprising a plurality of containers, each container comprising at least one cell with an increased frequency of HDR-mediated genome modification. In one embodiment, the present disclosure provides an arrangement of cells with HDR-promoting agents and, optionally, genome-editing molecules, wherein the cells are in an array format (e.g., in a multi-well plate), encapsulated or enclosed in vesicles, liposomes, or droplets (available, e.g., in a microfluidic device), or discretely attached to a matrix or discrete particles or beads; a specific embodiment is such an arrangement of a plurality of cells with increased frequency of HDR-mediated genome modification provided in an array format, further comprising at least one genome-editing molecule (e.g., an RNA-guided DNA nuclease, at least one guide RNA, or a ribonucleoprotein comprising both an RNA-guided DNA nuclease and at least one guide RNA) (which can be different for at least some positions on the array or even for each position on the array), and optionally at least one chemical, enzymatic, or physical delivery agent.

[0067] In the systems and methods provided herein, eukaryotic cells (e.g., plant cells) can be exposed to one or more HDR promoters and / or one or more gene editing molecules in any time sequence. In certain embodiments, HDR promoters and gene editing molecules are provided simultaneously. In other embodiments, genome editing molecules are provided after providing an HDR promoter. In other embodiments, gene editing molecules are provided before providing an HDR promoter. In short, HDR promoters can be provided to eukaryotic cells (e.g., plant cells) before, simultaneously with, or after the cells are exposed to gene editing molecules.

[0068] Provided herein are eukaryotic cells (e.g., plant cells) with an increased frequency of genome modification mediated by homology-directed repair (HDR) imparted by HDR accelerators (e.g., SSAPs, exonucleases, and SSBs) and / or modified DNA donor templates. The disclosure also provides compositions derived from or grown from plant cells or plant protoplasts with an increased frequency of genome modification mediated by HDR provided by the systems and methods disclosed herein; such compositions include multiple protoplasts or cells, calli, somatic embryos, somatic meristems, embryogenic calli, or regenerated plants grown from plant cells or plant protoplasts with an increased frequency of genome modification mediated by HDR. The frequency of genome modification mediated by HDR increased in cells subjected to HDR accelerators and / or modified DNA donor templates can be assessed by a variety of techniques. In certain embodiments, such techniques can compare the HDR frequency observed in cells subjected to HDR accelerators with the HDR frequency in control cells that have not been subjected to HDR accelerators (e.g., SSAPs, exonucleases, and SSBs) and / or modified DNA donor templates.

[0069] In certain embodiments, the eukaryotic cells (e.g., plant cells) used in the systems, methods, and compositions provided herein may include non-dividing cells. Such non-dividing cells may include plant cell protoplasts, eukaryotic cells subjected to one or more genetic and / or drug-induced cell cycle blocks, etc. In certain embodiments, after treatment with HDR promoters (e.g., SSAP, exonucleases, and SSB) and / or gene editing molecules (which may optionally include a modified DNA donor template provided herein), non-dividing cells may be induced to divide (e.g., by reversing or eliminating genetic or drug cell cycle blocks).

[0070] In certain embodiments, the eukaryotic cells (e.g., plant cells) used in the systems, methods, and compositions provided herein may include cleaving cells. cleaving cells may include those found in various plant tissues including leaves, meristems, and embryos. These tissues include, but are not limited to, cleaving cells from young leaves, meristems, and scutellum tissues of maize (from embryos about 8 or 10 to about 12 or 14 days after pollination (DAP). The isolation of maize embryos has been described in several publications (Brettschneider, Becker, and 1997; Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments, basal leaf tissue (e.g., leaf tissue located approximately 0 to 3 cm from the ligule of a maize plant; Kirienko, Luo, and Sylvester 2012) is targeted for HDR-mediated gene editing. Methods for obtaining regenerative plant structures and regenerated plants from HDR-mediated plant cell gene editing provided herein can be adapted from the methods disclosed in U.S. Patent Application Publication No. 20170121722 (incorporated herein by reference in its entirety and particularly with respect to such disclosures). In certain embodiments, a single plant cell subjected to HDR-mediated gene editing will produce a single regenerative plant structure. In certain embodiments, a single regenerative plant cell structure can be formed from a single cell on or within an explant that has undergone HDR-mediated gene editing. vii. Plant regeneration

[0071] In certain embodiments, the method provided herein may include growing or regenerating plants from plant cells or from regenerable plant structures derived from the plant cells for gene editing of improved HDR mediation. In certain embodiments, plants may further comprise the transgenic, target gene editing or genome editing of the insertion provided by the methods and compositions disclosed herein. In certain embodiments, callus is produced by plant cells, and plantlets and plants are produced by such callus. In other embodiments, whole seedlings or plants are grown directly from plant cells without the callus stage. Therefore, other related aspects relate to whole seedlings and plants grown or regenerated from plant cells or plant protoplasts with target gene editing or genome editing, and the seeds of such plants. In certain embodiments in which plant cells or plant protoplasts are subjected to genetic modification (for example, genome editing is performed by DNA nucleases such as RNA guidance), the plants grown or regenerated show a phenotype related to genetic modification. In certain embodiments, the plants grown or regenerated include two or more hereditary or epigenetic modifications in their genome, which provide at least one purpose phenotype in combination. In certain embodiments, a heterogeneous population of plant cells with a targeted gene edit or genome edit, at least some of which include at least one genetic or epigenetic modification, is provided by the method; related aspects include plants with a desired phenotype associated with the genetic or epigenetic modification, provided by: regenerating a plant with the desired phenotype from a plant cell or plant protoplast selected from a heterogeneous population of plant cells with a targeted gene edit or genome edit, or selecting a plant with the desired phenotype from a heterogeneous population of plants grown or regenerated from a population of plant cells with a targeted gene edit or genome edit. Examples of phenotypes of interest include herbicide resistance, improved tolerance to abiotic stresses (e.g., tolerance to extreme temperatures, drought, or salt) or biotic stresses (e.g., resistance to nematodes, bacterial, or fungal pathogens), improved nutrient or water utilization, modified lipid, carbohydrate, or protein composition, improved flavor or appearance, improved storage characteristics (e.g., resistance to bruising, browning, or softening), increased yield, altered morphology (e.g., floral structure or color, plant height, branching, root structure). In one embodiment, a heterogeneous population of plant cells with a targeted gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) is exposed to conditions that allow expression of the desired phenotype; for example, selection for herbicide resistance can include exposing a population of plant cells with a targeted gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) to an amount of herbicide or other growth-inhibiting or toxic substance, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive the treatment.Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can be adapted from published procedures (Roest and Gilissen, Acta Bot. Neerl. [Netherlands Botanical Journal], 1989, 38(1), 1-23; Bhaskaran and Smith, Crop Sci. [Crop Science] 30(6): 1328-1337; Ikeuchi et al., Development [Development], 2016, 143: 1442-1451). Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can also be adapted from U.S. Patent Application Publication No. 20170121722 (incorporated herein by reference in its entirety and particularly with respect to such disclosure). Also provided are heterogeneous groups, arrays or libraries of such plants, offspring or seeds of such plants grown or regenerated from plant cells or plant protoplasts with target gene editing or genome editing, parts of plants (including plant parts grafted as scions or rootstocks), products made from plants or their seeds (e.g., fruits or other edible plant parts, clean grains or seeds, edible oils, flour or starch, proteins and other processed products). Embodiments include plants grown or regenerated from plant cells with target gene editing or genome editing, wherein these plants comprise cells or tissues that do not have genetic or epigenetic modifications, for example, grafted plants wherein the scion or rootstock contains genetic or epigenetic modifications, or chimeric plants wherein some but not all cells or tissues contain genetic or epigenetic modifications. Plants that are commonly useful for grafting include many fruit trees and plants, such as many citrus trees, apples, stone fruits (e.g., peaches, apricots, cherries and plums), avocados, tomatoes, eggplants, cucumbers, melons, watermelons, grapes and various ornamental plants, such as roses. Grafted plants can be grafts between the same or different (usually related) species. Other related aspects include hybrid plants provided by hybridizing a first plant grown or regenerated from a plant cell or plant protoplast with target gene editing or genome editing and with at least one genetic or epigenetic modification with a second plant, wherein the hybrid plant comprises genetic or epigenetic modification; seeds produced by hybrid plants are also considered. Related aspects are also envisioned to be offspring seeds and offspring plants, including hybrid seeds and hybrid plants with regenerated plants as parents or ancestors. The plant cells and derived plants and seeds disclosed herein can be used for various purposes useful to consumers or growers. The complete plant itself may be desirable, for example, as a plant grown as a cover crop or ornamental plant. In other embodiments, processed products are made from plants or their seeds, such as extracted proteins, oils, sugars and starches, fermented products, animal feed or human food, wood and wood products, pharmaceuticals and various industrial products. viii. Providing HDR Promoters to Eukaryotic Cells

[0072] The SSAP, exonuclease, and / or SSB that increases the frequency of HDR can be provided to a eukaryotic cell (e.g., a plant cell or plant protoplast) by any suitable technique. In certain embodiments, the SSAP, exonuclease, and / or SSB is provided by directly contacting the cell with the SSAP, exonuclease, and / or SSB, or a polynucleotide encoding the SSAP, exonuclease, and / or SSB. In certain embodiments, the SSAP, exonuclease, and / or SSB is provided by transporting the SSAP, exonuclease, and / or SSB, or a polynucleotide encoding the SSAP, exonuclease, and / or SSB, into the cell using a chemical, enzymatic, or physical agent. In certain embodiments, the SSAP, exonuclease and / or SSB is provided by bacterial (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.)-mediated transfection of plant cells or plant protoplasts with a polynucleotide encoding the SSAP, exonuclease and / or SSB; see, e.g., Broothaerts et al. (2005) Nature, 433:629-633. In one embodiment, the SSAP, exonuclease, and / or SSB are provided by transcribing DNA encoding the SSAP, exonuclease, and / or SSB in a plant cell or plant protoplast and stably integrating it into the plant cell genome, or provided to the plant cell or plant protoplast in the form of a plasmid or expression vector (e.g., a viral vector) encoding the SSAP, exonuclease, and / or SSB. In certain embodiments, the SSAP, exonuclease, and / or SSB are provided to the plant cell or plant protoplast as a polynucleotide encoding the SSAP, exonuclease, and / or SSB (e.g., in the form of an RNA encoding the SSAP, exonuclease, and / or SSB (e.g., an mRNA or RNA containing an internal ribosome entry site (IRES))). In certain embodiments, the SSAP, exonuclease and / or SSB are provided to a plant cell or plant protoplast as a polynucleotide encoding a polyprotein comprising the SSAP, exonuclease and / or SSB in any order, the amino acid sequence of which comprises a protease recognition site or a self-processing protein sequence inserted between the encoded SSAP, exonuclease and / or SSB.Examples of such protease recognition sequences include the spacer region of plant metallothionein-like protein (PsMTa), which can be cleaved by endogenous plant proteases (Unwin et al., 1998) or the recognition sequence of a specific protease (e.g., TVMV Nia protease; Dasgupta et al., 1998) (which is also provided in the cell). Examples of such self-processing protein sequences include the foot-and-mouth disease virus (FMDV) 2A sequence (SEQ ID NO: 33; Halpin, C. et al., 1999). Genome editing molecules can also be introduced into plant cells by similar techniques. ix. Transient Expression of HDR Promoters

[0073] In certain embodiments of the methods, systems, cells and compositions provided herein, transient expression of HDR promoters and / or genome editing molecules is used. Transient expression of SSAPs, exonucleases and / or SSBs (which increase HDR frequency) or genome editing molecules can be achieved by a variety of techniques. In some embodiments, the expression of HDR promoters is inducible. In certain embodiments, SSAPs, exonucleases, SSBs and / or genome editing molecules are provided directly to cells, systems, methods and compositions as isolated molecules, as isolated products or semi-purified products of a cell-free synthesis process (e.g., in vitro translation), or as isolated products or semi-purified products in a cell-based synthesis process (e.g., such as in bacteria or other cell lysates). In certain embodiments, SSAPs, exonucleases, SSBs and / or genome editing molecules are targeted to cells or nuclei in a manner to ensure transient expression (e.g., by methods adapted from Gao et al. 2016; or Li et al. 2009). In certain embodiments, SSAP, exonuclease, SSB and / or genome editing molecules are delivered to cells by delivering SSAP, exonuclease, SSB and / or genome editing molecules in the absence of any polynucleotides encoding SSAP, exonuclease, SSB and / or genome editing molecules. Examples of exogenous agents that can be delivered in the absence of any encoding polynucleotides include SSAP, exonuclease, SSB, sequence-specific endonuclease and RNA guides. RNA-guided DNA-binding polypeptides / RNA guides can be delivered separately and / or as RNP complexes. In certain embodiments, SSAP, exonuclease and / or SSB proteins can be produced in a heterologous system, purified and delivered to plant cells by particle bombardment (e.g., by a method adapted from Martin-Ortigosa and Wang 2014). In embodiments where SSAP, exonuclease and / or SSB are delivered in the absence of any encoding polynucleotides, it is contemplated that the delivered agent degrades over time to result in transient expression in the absence of sustained expression of any introduced encoding polynucleotides. In certain embodiments, the SSAP, exonuclease, and / or SSB are delivered to cells by delivering a polynucleotide encoding the SSAP, exonuclease, and / or SSB. In certain embodiments, the SSAP, exonuclease, and / or SSB can be encoded on a bacterial plasmid and delivered to plant tissues via particle bombardment (e.g., by methods adapted from Hamada et al. 2018; or Kirienko, Luo, and Sylvester 2012). In certain embodiments, the SSAP, exonuclease, and / or SSB can be encoded on T-DNA and transiently transferred into plant cells using Agrobacterium (e.g., by methods adapted from Leonelli et al. 2016; or Wu et al. 2014).In certain embodiments, the SSAP, exonuclease, and / or SSB can be encoded in a viral genome and delivered to a plant (e.g., by methods adapted from Honig et al. 2015). In certain embodiments, the SSAP, exonuclease, and / or SSB can be encoded in an mRNA or RNA comprising an IRES and delivered to a target cell. In certain embodiments where the SSAP, exonuclease, and / or SSB comprises an RNA-guided DNA-binding polypeptide and an RNA guide, the polypeptide or guide can be delivered by a combination of: (i) an encoding polynucleotide for the polypeptide or guide; and (ii) the polypeptide or guide itself in the absence of the encoding polynucleotide. In certain embodiments, the SSAP, exonuclease, and / or SSB is delivered to a plant cell by delivering a polynucleotide encoding an HDR promoter. In certain embodiments, the polynucleotide encoding the SSAP, exonuclease and / or SSB is not integrated into the plant cell genome (e.g., as a polynucleotide lacking a sequence providing integration, agroinfiltration of an integration-defective T-DNA vector or system, or in a viral vector), is not operably linked to a polynucleotide providing autonomous replication, and / or has only factors providing autonomous replication (e.g., viral replication proteins). Suitable techniques for transient expression (including biolistic and other delivery of polynucleotides, agroinfiltration, and the use of viral vectors disclosed by Canto, 2016 and others) can be applied to transient expression of the SSAP, exonuclease, and / or SSB provided herein. Transient expression of the agent encoded by the non-integrated polynucleotide is achieved by excision of the polynucleotide and / or regulated expression of the agent. In certain embodiments, the polynucleotide encoding the SSAP, exonuclease, and / or SSB is integrated into the eukaryotic cell genome (e.g., a plant nuclear or plastid genome), and transient expression of the agent is achieved by excision of the polynucleotide and / or regulated expression of the SSAP, exonuclease, and / or SSB. The polynucleotide encoding the agent can be excised using a site-specific recombination system (e.g., Cre-Lox, FLP-FRT). Regulated expression of the agent can be achieved by methods including: (i) operable connection of a polynucleotide encoding the agent to a developmentally regulated, de-repressed, and / or inducible promoter; and / or (ii) introduction of a polynucleotide (e.g., dsRNA or miRNA) that induces siRNA-mediated agent inhibition. Suitable site-specific recombination systems and developmentally regulated, de-repressed, and / or inducible promoters include those disclosed in U.S. Patent Application Publication No. 20170121722 (incorporated herein by reference in its entirety and particularly with respect to such disclosures).

[0074] Polynucleotides that can be used to achieve transient expression of SSAPs, exonucleases, SSBs and / or genome editing molecules (e.g., polynucleotides encoding SSAPs, exonucleases, SSBs, sequence-specific endonucleases, RNA-guided endonucleases and / or guide RNAs) include: (a) double-stranded RNA; (b) single-stranded RNA; (c) chemically modified RNA; (d) double-stranded DNA; (e) single-stranded DNA; (f) chemically modified DNA; or (g) a combination of (a)-(f). Certain embodiments of the polynucleotides further include additional nucleotide sequences that provide useful functions; non-limiting examples of such additional nucleotide sequences include aptamer or riboswitch sequences, nucleotide sequences that provide secondary structures (e.g., stem loops) or sequence-specific sites for enzymes (e.g., sequence-specific recombinase or endonuclease sites), T-DNA (e.g., DNA sequences encoding SSAPs, exonucleases, and / or SSBs are surrounded by left and right T-DNA borders from Agrobacterium species or from other bacteria that infect or induce plant tumors), DNA nuclear targeting sequences, regulatory sequences (e.g., promoter sequences), and transcript stabilization or destabilization sequences. Certain embodiments of the polynucleotides include those in which the polynucleotides are complexed or covalently or non-covalently bound to non-nucleic acid elements (e.g., carrier molecules, antibodies, antigens, viral movement proteins, cell-penetrating or pore-forming peptides, polymers, detectable labels, quantum dots, microparticles, or nanoparticles). In some embodiments, one or more components provided herein are transiently expressed by induction of an inducible promoter. x. Delivery of HDR promoters

[0075] Various treatments can be used to deliver gene editing molecules and / or SSAPs, exonucleases, and / or SSBs that increase the frequency of HDR to eukaryotic cells (e.g., plant cells). In certain embodiments, one or more treatments are used to deliver HDR promoters (e.g., comprising polynucleotides, polypeptides, or combinations thereof) to eukaryotic or plant cells, for example, through barriers such as cell walls, plasma membranes, nuclear envelopes, and / or other lipid bilayers. In certain embodiments, a composition comprising polynucleotides, polypeptides, or RNPs containing one or more agents is directly delivered, for example, by direct contact of the composition with a eukaryotic cell. The above-mentioned composition can be provided in the form of a liquid, solution, suspension, emulsion, reverse emulsion, colloid, dispersion, gel, liposome, micelle, injectable material, aerosol, solid, powder, microparticle, nanoparticle or a combination thereof, which can be directly applied to eukaryotic cells, eukaryotic tissues, eukaryotic organs, eukaryotic organisms, plants, plant parts, plant cells or plant explants (for example, by scraping or puncturing or otherwise disrupting the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, plant cells or plant protoplasts are immersed in a composition containing liquid SSAP, nuclease and / or SSB to deliver the agent to the plant cells. In certain embodiments, negative pressure or positive pressure is used, for example, using vacuum infiltration or applying fluid mechanics or fluid pressure to deliver the composition containing the agent. In certain embodiments, the composition containing the pharmaceutical agent is introduced into plant cells or plant protoplasts, for example, by microinjection or by rupture or deformation of the cell wall or cell membrane, for example, by physical treatment, such as by applying negative or positive pressure, shear force, or with a chemical or physical delivery agent (e.g., surfactant), liposome or nanoparticle treatment; see, for example, as described in U.S. Published Patent Application 2014 / 0287509 (incorporated herein in its entirety by reference), using microfluidic flow to deliver the material to the cell by cell deformation and contraction. Other techniques for delivering the composition containing the pharmaceutical agent to eukaryotic cells, plant cells or plant protoplasts include: ultrasound or sonication; vibration, friction, shear stress, eddy current, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or rupture; enzymatic cell wall or cell membrane rupture or permeabilization; abrasion or mechanical scratching (e.g., abrasion with emery or other granular abrasives or scratching with a file or sandpaper) or chemical scratching (e.g., treatment with an acid or caustic agent); and electroporation.In certain embodiments, the composition containing the agent is provided by bacterial (e.g., Agrobacterium species, Rhizobium species, Sinorhizobium species, Mesorhizobium species, Bradyrhizobium species, Azotobacter species, Leafbacterium species)-mediated transfection of plant cells or plant protoplasts with polynucleotides encoding the agent (e.g., SSAP, exonuclease, SSB, sequence-specific endonuclease, and / or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433: 629-633. Any one or a combination of these techniques may alternatively be used for plant explants, plant parts or tissues, or whole plants (or seeds) from which plant cells are optionally subsequently obtained or isolated; in certain embodiments, the composition containing the agent is delivered in a separate step after the plant cells are isolated. In certain embodiments, the above methods may also be used to introduce genome editing molecules into eukaryotic cells (e.g., plant cells).

[0076] In an embodiment, the treatment employed in delivering SSAPs, exonucleases, and / or SSBs that increase the frequency of HDR to eukaryotic cells (e.g., plant cells) is performed under a specific thermal regime, which may involve one or more suitable temperatures, such as chilling or cold stress (exposure to temperatures below that at which normal plant growth occurs), or heating or heat stress (exposure to temperatures above that at which normal plant growth occurs), or treatment at a combination of different temperatures. In certain embodiments, in one or more steps separate from the delivery of the agent, a specific thermal regime is performed on the plant cell or plant, plant explant, or plant part from which the plant cell or plant protoplast is subsequently obtained or isolated. In certain embodiments, the methods described above may also be used to introduce genome editing molecules into eukaryotic cells.

[0077] In certain embodiments of the plant parts, systems, methods and compositions provided herein, the whole plant or plant part or seed, or isolated plant cell, plant explant, or plant cell or plant protoplast from which the plant or plant part is obtained or isolated, treated with one or more delivery agents, which may include at least one chemical, enzymatic or physical agent, or a combination thereof. In certain embodiments, the SSAP, exonuclease and / or SSB that increases the frequency of HDR further comprises one or more than one chemical, enzymatic or physical agent for delivery. Treatment with a chemical, enzymatic or physical agent may be performed simultaneously with the delivery of the agent, or in one or more separate steps before or after the delivery of the agent. In certain embodiments, a chemical, enzymatic or physical agent or a combination of these is associated or complexed with a polynucleotide composition, a donor template polynucleotide, a SSAP, an exonuclease and / or a SSB; examples of such associations or complexes include those involving non-covalent interactions (e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles or other heterogeneous compositions) and covalent interactions (e.g., peptide bonds, bonds formed using cross-linking agents). In a non-limiting example, SSAP, exonuclease and / or SSB are provided as liposome complexes with cationic lipids; SSAP, exonuclease and / or SSB are provided as complexes with carbon nanotubes; and / or SSAP, exonuclease and / or SSB are provided as fusion proteins between agents and cell penetrating peptides. Examples of agents for delivering SSAP, exonuclease and / or SSB include various cationic liposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J. Controlled Release, 123: 1-10, and cross-linked multilamellar liposomes described in U.S. Patent Application Publication 2014 / 0356414A1 (incorporated herein by reference in its entirety). In any of the foregoing embodiments, it is further contemplated that the above method can also be used to introduce genome editing molecules into eukaryotic cells (e.g., plant cells).

[0078] In certain embodiments, chemical agents for delivering SSAPs, exonucleases, and / or SSB proteins or polynucleotides encoding the same that increase the frequency of HDR may include: (a) solvents (e.g., water, dimethyl sulfoxide, dimethylformamide, acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxane, polyethylene glycol, and other solvents that are miscible or emulsifiable with water, or other solvents that dissolve nucleotide phosphates in non-aqueous systems); (b) Fluorocarbons (e.g. perfluorodecalin, perfluoromethyldecalin); (c) diols or polyols (e.g. propylene glycol, polyethylene glycol); (d) surfactants, including cationic surfactants, anionic surfactants, nonionic surfactants and amphiphilic surfactants, such as alkyl or aryl sulfates, phosphates, sulfonates or carboxylates; primary, secondary or tertiary amines; quaternary ammonium salts; betaines; cationic lipids; phospholipids; primary tallow amines; bile acids, such as cholic acid; long chain alcohols; silicone surfactants, including nonionic silicone surfactants (such as trisiloxane ethoxylate surfactants) or silicone polyether copolymers, such as a copolymer of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxypolypropylene glycol methyl ether (commercially available as SILWET L-77 TM brand surfactant, having CAS No. 27306-78-1 and EPA No. CAL.REG.NO. 5905-50073-AA, Momentive Performance Materials, Inc., Albany, New York); specific examples of useful surfactants include sodium lauryl sulfate, Tween series surfactants, Triton-X100, Triton-X114, CHAPS and CHAPSO, Tergitol type NP-40, Nonidet P-40; (e) lipids, lipoproteins, and lipopolysaccharides; (f) acids, alkalis and caustic agents; (g) peptides, proteins, or enzymes (e.g., cellulases, pectinases, maceroenzymes, pectinases), including cell-penetrating or pore-forming peptides (e.g., (BO100)2K8, Genscript); polylysine, polyarginine, or polyhomoarginine peptides; gamma-zein, see U.S. Patent Application Publication No. 2011 / 0247100, incorporated herein by reference in its entirety; transcriptional activators of human immunodeficiency virus type 1 ("HIV-1 Tat") and other Tat proteins, see, e.g., www[dot]lifetein[dot]com / Cell_Penetrating_Peptides[dot]html and (2012) Mol. Therapy - Nucleic Acids, 1:e27, 1-17); octaarginine or nonaarginine; polyhomoarginine (see Unnamalai et al. (2004) FEBS Letters, 566:307-310); see also the cell-penetrating peptide CPPsite 2.0 database, publicly available at crdd[dot]osdd[dot]net / raghava / cppsite / (h) RNase inhibitors; (i) cationic branched or linear polymers, such as chitosan, polylysine, DEAE-dextran, polyvinylpyrrolidone ("PVP"), or polyethyleneimine ("PEI," e.g., PEI, branched, MW 25,000, CAS#9002-98-6; PEI, linear, MW 5000, CAS#9002-98-6; PEI linear, MW 2500, CAS#9002-98-6); (j) dendrimers (see, e.g., U.S. Patent Application Publication No. 2011 / 0093982, herein incorporated by reference in its entirety); (k) counterions, amines or polyamines (e.g., spermine, spermidine, putrescine), osmolytes, buffers and salts (e.g., calcium phosphate, ammonium phosphate); (l) polynucleotides (e.g., nonspecific double-stranded DNA, salmon sperm DNA); (m) Transfection agents (e.g., and as well as (all from Thermo Fisher Scientific, Waltham, MA), PepFect (see Ezzat et al. (2011) Nucleic Acids Res., 39:5284-5298), Transfection reagent (Mirus Bio, LLC, Madison, Wisconsin) and polylysine, polyhomoarginine, and polyarginine molecules, including octaarginine and nonaarginine, as described by Lu et al. (2010) J. Agric. Food Chem., 58:2288-2294; (n) antibiotics, including nonspecific DNA double-strand break inducers (e.g., phleomycin, bleomycin, talimycin); and / or (o) Antioxidants (e.g., glutathione, dithiothreitol, ascorbic acid).

[0079] In any of the foregoing embodiments, it is further contemplated that the above-described chemical reagents can also be used to introduce genome-editing molecules into eukaryotic cells (e.g., plant cells).

[0080] In certain embodiments, a chemical agent is provided simultaneously with the SSAP, exonuclease, and / or SSB that increases the frequency of HDR. In certain embodiments, the SSAP, exonuclease, and / or SSB are covalently or non-covalently linked or compounded with one or more chemical agents; for example, the SSAP, exonuclease, SSB, and / or sequence-specific endonuclease can be covalently linked to a peptide or protein (e.g., a cell penetrating peptide or a pore-forming peptide) or non-covalently linked to a cationic lipid, a polycation (e.g., a polyamine), or a cationic polymer (e.g., PEI). In certain embodiments, the SSAP, exonuclease, and / or SSB are compounded with one or more chemical agents to form, for example, a solution, a liposome, a micelle, an emulsion, an inverted emulsion, a suspension, a colloid, or a gel. In any of the above embodiments, it is further contemplated that genome editing molecules comprising polynucleotides and / or polypeptides can also be delivered as described above.

[0081] In certain embodiments, the physical agent used to deliver SSAPs, exonucleases, and / or SSBs (which increase HDR frequency) is at least one selected from the group consisting of particles or nanoparticles of various size ranges and shapes (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics), magnetic particles or nanoparticles (e.g., silenceMag Magnetotransfection TMIn some embodiments, microparticles and nanoparticles can be used to deliver SSAPs, exonucleases, and / or SSBs. Useful microparticles and nanoparticles include those made from metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomposites, or nanotubes), and composites (e.g., polyvinylcarbazole / graphene, polystyrene / graphene, platinum / graphene, palladium / graphene nanocomposites). In certain embodiments, such microparticles and nanoparticles are further covalently or non-covalently functionalized, or further include a modifier or cross-linking material, such as a polymer (e.g., linear or branched polyethyleneimine, polylysine), a polynucleotide (e.g., DNA or RNA), a polysaccharide, a lipid, a polyglycol (e.g., polyethylene glycol, thiolated polyethylene glycol), a polypeptide or protein, and a detectable label (e.g., a fluorophore, an antigen, an antibody, or a quantum dot). In various embodiments, such microparticles and nanoparticles are neutral, positively charged, or negatively charged. Embodiments of compositions comprising microparticles include those formulated as, for example, liquids, colloids, dispersions, suspensions, aerosols, gels, and solids. Embodiments include nanoparticles fixed to a surface or support, for example, an array of carbon nanotubes arranged vertically on a silicon or copper wafer substrate. Embodiments include polynucleotide compositions comprising microparticles (e.g., gold or tungsten or magnetic particles) delivered by biolistic techniques or using magnetic forces. Particle sizes used in biolistics are typically in the "microparticle" range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micron size range (see, e.g., Gene Gun System, Bio-Rad, Hercules, CA, Instruction Manual; Randolph-Anderson et al. (2015) “Sub-micron gold particles are superior to larger particles for efficient transformation of organelles andsome cell types Transformation, submicron gold particles outperform larger particles [], Bio-Rad US / EG Brief 2015), but successful biolistic delivery has been reported in cultured animal cells using larger (40 nm) nanoparticles; see O'Brian and Lummis (2011) BMC Biotechnol., 11:66-71. Other examples of useful particles are nanoparticles, which are generally in the nanometer (nm) size range or less than 1 micron, for example, having a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm. Commercially available nanoparticles (all from Sigma-Aldrich) Corp.), St. Louis, MO) include gold nanoparticles having a diameter of 5, 10, or 15 nm; silver nanoparticles having a particle size of 10, 20, 40, 60, or 100 nm; palladium "nanopowders" having a particle size of less than 25 nm; single-walled, double-walled, and multi-walled carbon nanotubes, e.g., having a diameter of 0.7-1.1, 1.3-2.3, 0.7-0.9, or 0.7-1.3 nm, or nanotube bundles having a size of 2-10 nm x 1-5 microns, 6-9 nm x 5 microns, 7-15 nm x 0.5-10 microns, 7-12 nm x 0.5-10 microns, 110-170 nm x 5-9 microns, 6-13 nm x 10 microns, or 110-170 nm x 5-9 microns. 2.5-20 microns. In certain embodiments, physical agents for delivering SSAPs, exonucleases, and / or SSBs may include materials such as gold, silicon, cerium, or carbon (e.g., gold or gold-coated nanoparticles), silicon carbide whiskers, corundum, porous silica nanoparticles, gelatin / silica nanoparticles, cerium oxide nanoparticles (nanoceria), or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs), such as single-, double-, or multi-walled carbon nanotubes and chemically functionalized forms thereof (e.g., carbon nanotubes functionalized with amide, amino, carboxylic acid, sulfonic acid, or polyethylene glycol moieties), and graphene or graphene oxide or graphene composites.Such physical agents that may be suitable for delivering SSAPs, exonucleases, and / or SSBs include those disclosed in: Wong et al. (2016) Nano Lett., 16: 1161-1172; Giraldo et al. (2014) Nature Materials, 13: 400-409; Shen et al. (2012) Theranostics, 2: 283-294; Kim et al. (2011) Bioconjugate Chem., 22: 2558-2567; Wang et al. (2010) J. Am. Chem. Soc. Comm., 132: 9274-9276; Zhao et al. (2016) Nanoscale Res. Lett. [Nanoscale Research Express], 11: 195-203; and Choi et al. (2016) J. Controlled Release [Controlled Release Journal], 235: 222-235. See also, for example, various types of particles and nanoparticles, their preparation and methods of use (e.g., in delivering polynucleotides and polypeptides to cells) disclosed in U.S. Patent Application Publication Nos. 2010 / 0311168, 2012 / 0023619, 2012 / 0244569, 2013 / 0145488, 2013 / 0185823, 2014 / 0096284, 2015 / 0040268, 2015 / 0047074 and 2015 / 0208663, all of which are incorporated herein by reference in their entirety. In any of the above embodiments, it is further contemplated that genome editing molecules comprising polynucleotides and / or polypeptides can also be delivered as described above.

[0082] In some embodiments, as used herein, "providing" includes bringing the components in the nucleus together. In some embodiments, the provision of one or more components is in the form of polypeptide delivery. In some embodiments, the delivery of one or more components is in the form of a polypeptide complexed by a polynucleotide. In some embodiments, the delivery of one or more components is in the form of ribonucleoprotein (RNP). In some embodiments, Cas and guide RNA are delivered as ribonucleoproteins. In some embodiments, RNP is delivered to cells using lipofection or electroporation. In some embodiments, polypeptide or RNP is delivered to cells by biolistics. In some embodiments, polypeptide or RNP is delivered to cells by PEG-mediated transfection. In some embodiments, components are delivered by sexual hybridization.

[0083] In some embodiments, the component is provided as RNA or DNA. For example, in some embodiments, one or more components are provided as mRNA. In some embodiments, the mRNA encodes a protein as one of the components. In some embodiments, the mRNA is translated in a cell to produce one or more components.

[0084] In some embodiments, one or more components are provided as nucleic acids that are integrated into a chromosome.

[0085] In some embodiments, i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) one or more of a single-stranded DNA binding protein (SSB) are provided by a progenitor cell comprising one or more of i)-v). In some embodiments, the progenitor cell is any cell described herein, e.g., a plant, animal, fungus, or other eukaryotic cell. In some embodiments, the progenitor cell does not comprise at least one of a sequence-specific endonuclease, a donor template DNA molecule, a SSAP, an exonuclease, and an SSB protein. In some embodiments, at least one of a sequence-specific endonuclease, a donor template DNA molecule, a SSAP, an exonuclease, and an SSB protein that the progenitor cell does not comprise is subsequently provided by delivering a polypeptide, DNA, or mRNA to the progenitor cell and / or sexual hybridization of the progenitor cell. In some embodiments, the components shown in Table A below are provided. Table A: Combinations of components provided by progenitor cells or by delivery and / or sexual crosses of progenitor cells xi. Gene-editing molecules

[0086] In certain embodiments in which the gene editing molecule is included in a gRNA (or a polynucleotide encoding a gRNA) provided in the composition, the composition further comprises a nuclease activity-deficient RNA-guided DNA binding polypeptide (or a polynucleotide encoding the same), and one or more chemical, enzymatic or physical agents may be used similarly. In certain embodiments, the RNA guide and the nuclease activity-deficient RNA-guided DNA binding polypeptide (ndRGDBP) or the polynucleotide encoding the same are provided separately, for example, in separate compositions. Such compositions may include other chemical or physical agents (e.g., solvents, surfactants, proteins or enzymes, transfection agents, microparticles or nanoparticles), such as those described above that can be used in polynucleotide compositions. For example, porous silica nanoparticles can be used to deliver DNA recombinases into maize cells; see, for example, Martin-Ortigosa et al. (2015) Plant Physiol. [Plant Physiology], 164: 537-547, and can be suitable for providing ndRGDBP or its polynucleotide encoding into maize or other plant cells. In one embodiment, the polynucleotide composition includes gRNA and ndRGDBP, and further includes a surfactant and a cell penetrating peptide (CPP) that can be operably connected to ndRGDBP. In one embodiment, the polynucleotide composition includes a plasmid or viral vector encoding gRNA and ndRGDBP, and further includes a surfactant and a carbon nanotube. In one embodiment, the polynucleotide composition includes multiple gRNAs and mRNA encoding ndRGDBP, and further includes particles (e.g., gold or tungsten particles), and the polynucleotide composition is delivered to plant cells or plant protoplasts by biolistics. In any of the above embodiments, it is further contemplated that other polynucleotides of interest including genome editing molecules can also be delivered before, during, or after the delivery of gRNA and ndRGDBP.

[0087] In certain embodiments, in the process of obtaining, separating or treating plant cells, the plant, plant explant or plant part from which the plant cell is obtained or separated is treated with one or more chemical, enzyme or physical reagents. In certain embodiments, the plant cell, plant, plant explant or plant part is treated with an abrasive, a caustic agent, a surfactant (e.g., Silwet L-77) or a cationic lipid or an enzyme (e.g., a cellulase). In any of the above embodiments, it is further contemplated that other polynucleotides of interest including genome editing molecules can also be delivered before, during or after the delivery of the HDR promoter.

[0088] In certain embodiments, one or more chemical, enzymatic or physical agents, alone or in combination with a polynucleotide composition encoding an SSAP, exonuclease and / or SSB that increases the frequency of HDR, are provided / applied to a location on a plant or plant part that is different from the location, part or tissue of the plant from which the plant cells are treated, obtained or isolated. In certain embodiments, the polynucleotide composition is applied to adjacent or distal cells or tissues and transported (e.g., via the vasculature or by intercellular movement) to the meristem, from which the plant cells are subsequently separated. In certain embodiments, the polynucleotide-containing composition is applied by soaking seeds or seed fragments or zygotes or somatic embryos in a polynucleotide-containing composition, thereby delivering the polynucleotides to the plant cells. In certain embodiments, a flower bud or stem tip is contacted with a polynucleotide-containing composition, thereby delivering the polynucleotides to cells in the flower bud or stem tip, from which the desired plant cells are obtained. In certain embodiments, a polynucleotide-containing composition is applied to the surface of a plant or plant part (e.g., a leaf surface), thereby delivering one or more polynucleotides to the tissues of the plant, from which the desired plant cells are obtained. In certain embodiments, whole plants or plant tissues are subjected to particle or nanoparticle-mediated delivery (e.g., biolistic or carbon nanotube or nanoparticle delivery) of a composition containing polynucleotides, thereby delivering one or more polynucleotides to cells or tissues, from which plant cells are subsequently obtained. In any of the above embodiments, it is further contemplated that other polynucleotides of interest, including genome editing molecules, may also be delivered before, during, or after the delivery of the HDR accelerator.

[0089] Genome editing molecules include gene editing molecules for inducing gene modification in plant cells with increased HDR-mediated genome modification frequency provided herein. In certain embodiments, such genome editing molecules may include: (i) a polynucleotide selected from the group consisting of an RNA guide for an RNA-guided nuclease, a DNA encoding an RNA guide for an RNA-guided nuclease; (ii) a nuclease selected from the group consisting of an RNA-guided nuclease, an RNA-guided DNA endonuclease, a type II Cas nuclease, Cas9, nCas9, a type V Cas nuclease, Cas12a, nCas12a, CasY, CasX, Cas12b, Cas12c, Cas12i, Cas14, an engineered nuclease, a codon-optimized nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease; (iii) a polynucleotide encoding one or more nucleases capable of achieving site-specific cleavage of a target nucleotide sequence; and / or (iv) a donor template DNA molecule. In certain embodiments, at least one delivery agent is selected from the group consisting of: solvents, fluorocarbons, diols or polyols, surfactants; primary, secondary or tertiary amines and quaternary ammonium salts; silicone surfactants; lipids, lipoproteins, lipopolysaccharides; acids, bases, caustics; peptides, proteins or enzymes; cell penetrating peptides; RNAse inhibitors; cationic branched or linear polymers; dendrimers; counterions, amines or polyamines, osmolytes, buffers and salts; polynucleotides; transfection agents; antibiotics; chelating agents such as ammonium oxalate, EDTA, EGTA, cyclohexanediaminetetraacetic acid, non-specific DNA double-strand break inducers; and antioxidants; particles or nanoparticles, magnetic particles or nanoparticles, abrasives or scratching agents, needles or microneedles, matrices and meshes. In certain embodiments, the eukaryotic cells (e.g., plant cells), systems, methods, or compositions comprising the cells provided herein further include (a) at least one cell having at least one Cas9, nCas9, Cas12a, nCas12a, CasY, CasX, Cas12b, Cas12c, or Cas12i nuclease or nickase; (b) at least one guide RNA; and (c) optionally, at least one chemical, enzymatic, or physical delivery agent.

[0090] The gene editing molecules used in the cells, systems, methods, compositions and reaction mixtures provided herein include molecules capable of introducing a double-strand break ("DSB") in double-stranded DNA (e.g., in genomic DNA or in a target gene located within genomic DNA) and an accompanying guide RNA or donor template polynucleotide. Examples of such gene editing molecules include: (a) a nuclease selected from the group consisting of: an RNA-guided nuclease, an RNA-guided DNA endonuclease, a type II Cas nuclease, Cas9, an nCas9 nickase, a type V Cas nuclease, a Cas12a nuclease, an nCas12a nickase, CasY, CasX, Cas12b, Cas12c, Cas12i, Cas14, an engineered nuclease, a codon-optimized nuclease, a zinc finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL effector nuclease) or nickase, an Argonaute, and a meganuclease or an engineered meganuclease; (b) a polynucleotide encoding one or more nucleases that are capable of effecting a site-specific change in the target editing site (e.g., introducing a DSB); (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an RNA-guided nuclease; and (d) a donor template polynucleotide.

[0091] CRISPR type genome editing can be applicable to eukaryotic cells (e.g., plant cells), systems, methods, and compositions provided herein in various ways. CRISPR elements, i.e., gene editing molecules comprising CRISPR endonucleases and CRISPR single guide RNAs or polynucleotides encoding them, can be used to achieve genome editing without the selective genetic markers that appear in the residues or offspring of the CRISPR elements. In certain embodiments, the CRISPR elements are provided directly to eukaryotic cells (e.g., plant bags), systems, methods, and compositions as isolated molecules, as isolated products or semi-purified products of a cell-free synthesis process (e.g., in vitro translation), or as isolated products or semi-purified products in a cell-based synthesis process (e.g., such as in bacteria or other cell lysates). In certain embodiments, the CRISPR elements of genome insertion can be used for plant lines applicable to systems, methods, and compositions provided herein. In certain embodiments, the plants or plant cells used in the systems, methods, and compositions provided herein may include a transgenic expressing a CRISPR endonuclease (e.g., Cas9, Cpf1 type, or other CRISPR endonucleases). In certain embodiments, one or more CRISPR nucleases with unique PAM recognition sites can be used. The guide RNA (sgRNA or crRNA and tracrRNA) forms an RNA-guided nuclease / guide RNA complex that can specifically bind to a sequence adjacent to the pre-spacer adjacent motif (PAM) sequence in the gDNA target editing site. The type of RNA-guided nuclease typically informs the location of a suitable PAM site and the design of the crRNA or sgRNA. G-rich PAM sites, such as 5'-NGG, are typically targeted for designing crRNA or sgRNA for use with Cas9 protein. T-rich PAM sites (e.g., 5'-TTTV[1], where "V" is A, C, or G) are typically targeted for designing crRNA or sgRNA for use with Cas12a protein (e.g., SEQ ID NOs: 27, 28, 29, and 30). Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1 (incorporated herein by reference for the disclosure of DNA encoding Cpf1 endonuclease and guide RNA and PAM site therein). Introducing one or more of a wide variety of CRISPR guide RNAs that interact with a CRISPR endonuclease that is integrated into the plant genome or otherwise provided to the plant can be used for gene editing to provide a desired phenotype or trait, trait screening, or gene editing-mediated trait introgression (e.g., for introducing a trait into a new genotype without backcrossing to the recurrent parent or with limited backcrossing to the recurrent parent).Multiple endonucleases can be provided in expression cassettes with appropriate promoters to allow multiplex genome editing in a spatially or temporally separated manner in chromosomal or episomal DNA.

[0092] CRISPR technology for editing genes in eukaryotic organisms is disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1, and U.S. Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. The Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1. Other CRISPR nucleases that can be used to edit the genome include Cas12b and Cas12c (see Shmakov et al. (2015) Mol. Cell, 60:385-397) and CasX and CasY (see Burstein et al. (2016) Nature, doi:10.1038 / nature21059). Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonucleases are disclosed in International Patent Application PCT / US2015 / 018104 (published as WO 2015 / 131101 and claiming priority to U.S. Provisional Patent Application 61 / 945,700). Methods for genome editing using CRISPR technology in plants are disclosed in U.S. patent application publications US 2015 / 0082478A1 and US2015 / 0059010 A1 and international patent application PCT / US 2015 / 038767A1 (published as WO 2016 / 007347 and claiming priority to U.S. provisional patent application 62 / 023,246). All patent publications cited in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, an RNA-guided endonuclease is used that leaves a blunt end after cleaving the target editing site at the endonuclease recognition sequence. Flat-end-cutting RNA-guided endonucleases include Cas9, Cas12c, and Cas12h (Yan et al., 2019). In certain embodiments, an RNA-guided endonuclease is used that leaves a staggered single-stranded DNA overhang after cleavage of the endonuclease recognition sequence. Staggered end-cleavage RNA-guided endonucleases include Cas12a, Cas12b, and Cas12e.

[0093] Methods, systems, compositions, eukaryotic cells (e.g., plant cells) can also use sequence-specific endonucleases or sequence-specific endonucleases and guide RNAs that cut single DNA strands at the endonuclease recognition sequence within the target editing site in dsDNA. This cutting of single DNA strands in dsDNA target editing sites is also referred to herein and elsewhere as "nicking" and can be achieved by various "nicking enzymes" or systems that provide nicking. Nicking enzymes that can be used include nCas9 (Cas9 comprising D10A amino acid substitutions), nCas12a (e.g., Cas12a comprising R1226A amino acid substitutions; Yamano et al., 2016), Cas12i (Yan et al. 2019), zinc finger nickases (e.g., Kim et al., disclosed in 2012), TALE nickases (e.g., Wu et al., disclosed in 2014), or combinations thereof. In certain embodiments, the system providing the nick may comprise a Cas nuclease (e.g., Cas9 and / or Cas12a) and a guide RNA molecule having at least one base mismatch with the DNA sequence in the target editing site (Fu et al., 2019). In certain embodiments, genomic modifications can be introduced into the target editing site by creating single-strand breaks (i.e., "nicks") at genomic locations that are no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 DNA base pairs apart. In certain illustrative and non-limiting embodiments, two nickases (i.e., CAS nucleases that introduce single-strand DNA breaks, including nCas9, nCas12a, Cas12i, zinc finger nickases, TALE nickases, combinations thereof, etc.) or nickase systems can guide nicking of nearby sites that are no more than about 10, 20, 30, 40, 50, 60, 80, or 100 DNA base pairs apart. In the case of using an RNA-guided nickase and an RNA guide, the RNA guide is adjacent to the PAM sequence in sufficient proximity (i.e., no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 DNA base pairs). In any of the foregoing embodiments using a nickase or nickase system, an exonuclease with 5' to 3' or 3' to 5' exonuclease activity that can recognize dsDNA substrates with internal breaks in one strand can be used. In certain embodiments, T7 phage exonuclease, E. coli exonuclease III, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 143 or 144 can be used in combination with a nickase or nickase system, SSAP, and SSB.

[0094] For the purpose of gene editing, CRISPR arrays can be designed to contain one or more guide RNA sequences corresponding to the desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. Cas9 requires at least 16 or 17 nucleotides of the gRNA sequence for DNA cleavage to occur; for Cpf1, at least 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage, and at least 18 nucleotides of the gRNA sequence are reported to be required for efficient DNA cleavage in vitro; see Zetsche et al. (2015) Cell, 163:759-771. In practice, guide RNA sequences are typically designed to have a length of 17-24 nucleotides (typically 19, 20, or 21 nucleotides) and precise complementarity (i.e., perfect base pairing) with the targeted gene or nucleic acid sequence; guide RNAs with less than 100% complementarity to the target sequence (e.g., gRNAs with a length of 20 nucleotides and 1-4 mismatches with the target sequence) can be used, but this can increase the likelihood of off-target effects. The design of effective guide RNAs for plant genome editing is disclosed in U.S. Patent Application Publication No. 2015 / 0082478A1 (the entire specification of which is incorporated herein by reference). Recently, efficient gene editing has been achieved using chimeric "single guide RNA" ("sgRNA"), an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and contains a tracrRNA (for binding a nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing); see, e.g., Cong et al. (2013) Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340. Chemically modified sgRNAs have been shown to be effective in genome editing; see, e.g., Hendel et al. (2015) Nature Biotechnol., 985-991. The design of efficient gRNAs for plant genome editing is disclosed in U.S. Patent Application Publication No. 2015 / 0082478A1 (the entire specification of which is incorporated herein by reference).

[0095] Other sequence-specific endonucleases capable of achieving site-specific modification of target nucleotide sequences in the systems, methods, and compositions provided herein include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL-effector nucleases or TALENs), Argonaute proteins, and meganucleases or engineered meganucleases. Zinc finger nucleases (ZFNs) are engineered proteins comprising a zinc finger DNA binding domain fused to a nucleic acid cleavage domain (e.g., a nuclease). The zinc finger binding domain provides specificity and can be engineered to specifically recognize any desired target DNA sequence. For a review of the construction and use of ZFNs in plants and other organisms, see, for example, Urnov et al. (2010) Nature Rev. Genet. [Nature Review Genetics], 11: 636-646. The zinc finger DNA binding domain is derived from the DNA binding domain of a large class of eukaryotic transcription factors, known as zinc finger proteins (ZFPs). The DNA binding domain of a ZFP typically comprises a tandem array of at least three zinc "fingers," each of which recognizes a specific DNA triplet. Many strategies can be used to design the binding specificity of zinc finger binding domains. A method called "modular assembly" relies on the functional autonomy of a single zinc finger with DNA. In this method, a given sequence is targeted by identifying the zinc fingers of each component triplet in the sequence and connecting them to a multi-finger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods aim to regulate the ability of zinc fingers to contact nucleotide bases outside of adjacent fingers and their target triplet. Generally, compared to naturally occurring zinc finger proteins, engineered zinc finger DNA binding domains have novel binding specificity. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, using a database of triplet (or quadruple) nucleotide sequences and single zinc finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more zinc finger amino acid sequences that bind to a specific triplet or quadruple sequence. See, for example, U.S. Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) are well known and described in the literature. In addition, enhancement of the binding specificity of zinc finger binding domains is described in U.S. Patent 6,794,136 (incorporated herein by reference in its entirety). In addition, any suitable linker sequence can be used to link the individual zinc finger domains together. Examples of linker sequences are well known, for example, see U.S. Patents 6,479,626; 6,903,185; and 7,153,949, which are incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fok1. Such endonucleases must dimerize to cleave DNA.Thus, Fok1 cleavage as part of a ZFN requires two adjacent and independent binding events, which must occur in the correct orientation and appropriate spacing to allow dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fok1 variants with enhanced activity have been described; see, for example, Guo et al. (2010) J. Mol. Biol., 400:96-107.

[0096] Transcription activator-like effectors (TALEs) are proteins secreted by certain Xanthomonas species that regulate gene expression in host plants and promote bacterial colonization and survival. TALEs act as transcription factors and regulate the expression of resistance genes in plants. Recent studies on TALEs have revealed the code that connects the repeat region of TALEs to their target DNA binding sites. TALEs contain highly conserved and repeated regions that consist of tandem repeats of fragments of mostly 33 or 34 amino acids. The repeat monomers differ from each other primarily at amino acid positions 12 and 13. A strong correlation has been found between the unique amino acid pairs at positions 12 and 13 and the corresponding nucleotides in the TALE binding site. The simple relationship between the amino acid sequence and the DNA recognition of the TALE binding domain allows the design of DNA binding domains with any desired specificity. TALEs can be linked to nonspecific DNA cleavage domains to prepare sequence-specific nucleases, called TAL effector nucleases or TALENs. As with ZFNs, restriction endonucleases such as Fok1 can be conveniently used. For a description of the use of TALENs in plants, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623-2628 and Mahfouz (2011) GM Crops, 2:99-103.

[0097] Argonaute is a protein that can function as a sequence-specific endonuclease by binding to a polynucleotide (e.g., a single-stranded DNA or single-stranded RNA comprising a sequence complementary to a target nucleotide sequence), which guides Argonaut to target a nucleotide sequence and achieve site-specific alteration of the target nucleotide sequence; see, e.g., U.S. Patent Application Publication No. 2015 / 0089681, incorporated herein by reference in its entirety.

[0098] In certain embodiments, endonuclease binds to an endonuclease recognition sequence. In certain embodiments, endonuclease cuts an endonuclease recognition sequence. In certain embodiments, term "endonuclease recognition sequence" is used interchangeably with endonuclease cleavage site sequence.

[0099] In some embodiments, no endonuclease is required. In some embodiments, the method is implemented by providing a compound that non-specifically introduces double-strand breaks. Exemplary double-strand break-inducing compounds (including hydroquinone (HQ), benzoquinone (BQ), pyrogallol (BT), hydrogen peroxide (H2O2), bleomycin (BLM), or sodium ascorbate (Vit C)) are used to introduce double-strand breaks.

[0100] The donor template DNA molecules used in the methods, systems, eukaryotic cells (e.g., plant cells) and compositions provided herein include DNA molecules comprising a first homology arm, a replacement DNA and a second homology arm from 5' to 3', wherein the homology arm comprises a sequence that is partially or completely homologous to a genomic DNA (gDNA) sequence flanking an endonuclease recognition sequence in the gDNA, and wherein the replacement DNA can comprise an insertion, deletion or substitution of one or more DNA base pairs relative to the target gDNA. In certain embodiments, the length of the donor DNA template homology arm can be from about 20, 50, 100, 200, 400 or 600 to about 800 or 1000 base pairs. In certain embodiments, the donor template DNA molecule can be delivered to a eukaryotic cell (e.g., a plant cell) as a circular (e.g., a plasmid or a viral vector including a geminivirus vector) or a linear DNA molecule. In certain embodiments, the circular or linear DNA molecule used may comprise a modified donor template DNA molecule comprising, from 5' to 3', a first copy of the endonuclease recognition sequence, a first homology arm, a replacement DNA, a second homology arm, and a second copy of the endonuclease recognition sequence. Without being limited by theory, such a modified DNA donor template molecule can be cleaved by the same sequence-specific endonuclease as the sequence-specific endonuclease used to cleave the endonuclease recognition sequence within the genomic DNA of the target editing site of the eukaryotic cell to release the donor template DNA molecule (which can participate in HDR-mediated genomic modification of the target editing site in the eukaryotic cell genome). In certain embodiments, the donor DNA template may comprise a linear DNA molecule comprising, from 5' to 3', a cut endonuclease recognition sequence, a first homology arm, a replacement DNA, a second homology arm, and a cut endonuclease recognition sequence. In certain embodiments, the cut endonuclease sequence may comprise the following: a blunt DNA end or a blunt DNA end that may optionally contain a 5' phosphate group. In certain embodiments, the endonuclease sequence of cutting comprises the DNA end with single-stranded 5 ' or 3 ' DNA overhang.The endonuclease recognition sequence of such cutting can be synthesized to produce by cutting complete target sequence or by the copy of the target sequence specific endonuclease recognition sequence of cutting.Donor DNA template can be chemically synthesized or enzymatically synthesized (for example, in polymerase chain reaction (PCR)).

[0101] It is also contemplated to use donor templates other than double-stranded DNA. For example, in some embodiments, a double-stranded DNA precursor is provided. In some embodiments, an RNA template for a reverse transcriptase is provided. In some embodiments, a reverse transcriptase is provided in addition to RNA. In some embodiments, the method includes using a single-stranded DNA donor template. In some embodiments, a single-stranded or double-stranded RNA template is used. In some embodiments, the method includes using a DNA / RNA hybrid. In some embodiments, a PNA is used to generate the donor template.

[0102] In some embodiments, more than one donor template is provided. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more donor templates are provided. In some embodiments, the donor templates target the same gene. In some embodiments, the donor templates target different genes in the same pathway. In some embodiments, the donor templates target multiple genes that perform the same function.

[0103] Other genome editing molecules for plant cells and methods provided herein can be used for plants or cells with transgenic or vectors comprising them. Such transgenic can confer useful traits, including herbicide tolerance, pest tolerance (for example, tolerance to insects, nematodes or plant pathogenic fungi and bacteria), improved yield, increased and / or quality-improved oil, starch and / or protein content, improved abiotic stress tolerance (for example, improved or enhanced water use efficiency or drought tolerance, osmotic stress tolerance, high salt stress tolerance, heat stress tolerance, enhanced cold tolerance, including cold germination tolerance), etc. Such transgenic includes transgenics that confer traits by exogenous protein expression and transgenics that confer traits by suppressing endogenous plant genes (for example, by inducing siRNA responses that suppress endogenous plant gene expression). U.S. Patent Application Publication Nos. 20170121722 and 20170275636 (incorporated herein by reference in their entirety and particularly with respect to such disclosures) disclose transgenics that can provide such traits.

[0104] In some embodiments, one or more polynucleotides or vectors (which drive the expression of one or more polynucleotides encoding any of the above-mentioned SSAPs, exonucleases and / or SSBs and / or genome editing molecules) are introduced into eukaryotic cells (e.g., plant cells). In certain embodiments, the polynucleotide vector comprises a regulatory element, such as a promoter operably linked to one or more polynucleotides encoding SSAPs, exonucleases and / or SSBs or genome editing molecules. In such embodiments, the expression of these polynucleotides can be controlled by selecting an appropriate promoter, particularly a promoter that is functional in eukaryotic cells (e.g., plant cells); useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., tissue-specific promoters, developmentally regulated promoters, or cell cycle-regulated promoters). Developmentally regulated promoters that can be used in plant cells include phospholipid transfer protein (PLTP), fructose-1,6-bisphosphatase protein, NAD(P)-binding Rossmann-Fold protein, adipocyte plasma membrane-associated protein-like protein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratory reduced 6 protein, D-glycerate 3-kinase, chloroplast-like protein, chlorophyll ab-binding protein 7, chloroplast-like protein, ultraviolet B inhibitory protein, Soul heme binding family protein, photosystem I reaction center subunit psi-N protein and short-chain dehydrogenase / reductase protein, as disclosed in U.S. Patent Application Publication No. 20170121722 (incorporated herein by reference in its entirety and specifically with respect to such disclosure). In certain embodiments, the promoter is operably linked to a nucleotide sequence encoding a plurality of guide RNAs, wherein the sequences encoding the guide RNAs are separated by cleavage sites (e.g., nucleotide sequences encoding microRNA recognition / cleavage sites or self-cleaving ribozymes) (see, e.g., Ferré-D'Amaré and Scott (2014) Cold Spring Harbor Perspectives Biol., 2: a003574). In certain embodiments, the promoter is an RNA polymerase III promoter operably linked to a nucleotide sequence encoding one or more guide RNAs. In certain embodiments, the promoter operably linked to one or more polynucleotides is a constitutive promoter that drives gene expression in a eukaryotic cell (e.g., a plant cell). In certain embodiments, the promoter drives gene expression in the nucleus or an organelle (e.g., a chloroplast or a mitochondria).Examples of constitutive promoters for plants include the CaMV 35S promoter disclosed in U.S. Patents 5,858,742 and 5,322,938, the rice actin promoter disclosed in U.S. Patent 5,641,876, the maize chloroplast aldolase promoter disclosed in U.S. Patent 7,151,204, and the nopaline synthase (NOS) and octopine synthase (OCS) promoters from Agrobacterium tumefaciens. In certain embodiments, the promoter operably linked to one or more polynucleotides encoding elements of the genome editing system is a promoter from fig mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter (which is active in photosynthetic tissue). Other promoters considered include cell-specific or tissue-specific or developmentally regulated promoters, for example, promoters that restrict expression of the nucleic acid targeting system to germline cells or germ cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline cells or germ cells). In certain embodiments, genomic alterations are limited to those cells from which DNA is inherited in subsequent generations, which is advantageous in the case of wishing to limit expression of the genome editing system to avoid genotoxicity or other unwanted effects. All patent publications cited in this paragraph are incorporated herein by reference in their entirety.

[0105] The expression vectors or polynucleotides provided herein may contain a DNA segment near the 3' end of the expression cassette that serves as a signal to terminate transcription and direct polyadenylation of the resulting mRNA, and may also support promoter activity. This 3' element is often referred to as a "3'-untranslated region" or "3'-UTR" or "polyadenylation signal." In some cases, the 3' element (or terminator) based on plant genes consists of a 3'-UTR and downstream non-transcribed sequences (Nuccio et al., 2015). Useful 3′ elements include the Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, and tr7 3′ elements disclosed in U.S. Patent No. 6,090,627 (incorporated herein by reference), and 3′ elements from plant genes (e.g., the heat shock protein 17, ubiquitin, and fructose-1,6-bisphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and β-tubulin genes from rice (Oryza sativa)) disclosed in U.S. Patent Application Publication 2002 / 0192813A1 (incorporated herein by reference).

[0106] In certain embodiments, the vector or polynucleotide comprising the expression cassette includes additional components, for example, a polynucleotide encoding a drug resistance or herbicide gene or a polynucleotide encoding a detectable marker, such as green fluorescent protein (GFP) or β-glucuronidase (GUS), to facilitate screening or selection of cells expressing the vector or polynucleotide. Selectable markers include genes that confer resistance to herbicidal compounds such as glyphosate, sulfonylureas, glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). Such selectable marker genes and selection agents include the maize HRA gene (Lee et al., 1988, EMBO J 7: 1241-1248), which confers resistance to sulfonylureas and imidazolinones; the CP4 gene, which confers resistance to glyphosate (U.S. Reissue Patent RE039247, specifically incorporated herein by reference in its entirety and with respect to such genes and related selection methods); the GAT gene, which confers resistance to glyphosate (Castle et al., 2004, Science 304: 1151-1154); genes that confer resistance to spectinomycin, such as the aadA gene (Svab et al., 1990, Plant Mol Biol. 14: 197-205); and the bar gene, which confers resistance to glufosinate (White et al., 1990, Nucl. Acids Res. Nucleic Acids Res. 25:1062); and PAT (or moPAT for maize, see Rasco-Gaunt et al., 2003, Plant Cell Rep. 21:569-76; see also Sivamani et al., 2019); and the PMI gene, which allows growth on mannose-containing media (Negrotto et al., 2000, Plant Cell Rep. 22:684-690).

[0107] In certain embodiments, counter-selection markers may be used in the eukaryotic cells (e.g., plants), methods, systems, and compositions provided herein. In certain embodiments, such counter-selection markers may be incorporated into any DNA at the target editing site that is not intended to be inserted into the host cell genome. In such embodiments, non-limiting examples of DNA with counter-selection markers include any DNA molecule connected to DNA encoding an HDR promoter (e.g., SSB, SSAP, and / or exonuclease), a gene editing molecule, and / or a donor template DNA molecule. A vector or DNA molecule comprising a donor template DNA molecule, wherein the counter-selection marker is connected to the donor template DNA and is optionally separated from the donor template DNA by a target editing site sequence. Examples of counter-selectable markers that can be used in plants include genes for cytosine deaminase (e.g., used in combination with 5-fluorocytosine; Schlaman and Hooykaas, 1997), phosphonate hydrolases (e.g., used in combination with phosphonates of glyphosate (including glycerol glyphosate); Dotson et al. 1996), and nitrate reductases (e.g., used in combination with chlorate on media containing ammonia as the sole nitrogen source; Nussaume et al. 1991).

[0108] In certain embodiments, the use of a selection marker is avoided by increasing the frequency of HDR provided by an HDR facilitator (i.e., SSAP, exonuclease, and / or SSB) and / or a modified template DNA molecule. In such embodiments, the selection marker and / or counter-selection marker can be omitted from any of the following: the donor template DNA molecule, the plasmid or any other vector (e.g., a viral vector) for delivering the donor template or other DNA molecule, or the polynucleotide used in the cells, systems, methods, or compositions provided herein. B. Genetic Engineering Methods

[0109] In one aspect, the present disclosure provides a method for genetic engineering of eukaryotic cells. In some embodiments, the method includes providing i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, the method includes delivering a nucleic acid encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

[0110] In another aspect, the present disclosure provides a method for genetically engineering a eukaryotic cell. In some embodiments, the method comprises i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), and iv) an exonuclease that at least partially converts a double-stranded DNA substrate into a single-stranded DNA product.

[0111] In another aspect, the method comprises i) a double-strand break-inducing compound, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), and iv) an exonuclease that can at least partially convert the double-stranded DNA substrate into a single-stranded DNA product. i. Genetic modification

[0112] Genetic engineering can be a reduction in gene function (i.e., activity in the encoded gene product). This may require a corresponding repair template, as discussed herein, to provide a defective sequence, or it can be by inducing DSBs. In particular, gene perturbation is gene knockdown. In some embodiments, the cell is a plant or animal cell. In some embodiments, genetic engineering is the introduction of a stop codon within a gene. In some embodiments, genetic engineering is a mutation in a promoter or start codon.

[0113] Alternatively, genetic engineering can be an increase in gene function (i.e., activity in the encoded gene product). This may require a corresponding repair template, as discussed herein, to provide a corrected sequence. In certain embodiments, genetic engineering is the replacement of one or more nucleotides in a protein-coding gene.

[0114] In some embodiments, the target editing site is located in a promoter region. In one embodiment, the nucleotide sequence can be a promoter, wherein editing of the promoter results in any one or any combination of the following: increased promoter activity, increased promoter tissue specificity, decreased promoter activity, decreased promoter tissue specificity, mutation of a DNA binding element, and / or deletion or addition of a DNA binding element.

[0115] In one embodiment, the nucleotide sequence can be a regulatory sequence in the cell genome. A regulatory sequence is a fragment of a nucleic acid molecule that can increase or decrease the expression of a specific gene in an organism. Examples of regulatory sequences include, but are not limited to, transcriptional activators, transcriptional repressors and translational repressors, splicing factors, miRNA, siRNA, artificial miRNA, CAAT box, CCAAT box, Pribnow box, TATA box, SECIS element, and polyadenylation signal. In certain embodiments, the editing of regulatory elements results in altered protein translation, RNA cutting, RNA splicing, or transcription termination.

[0116] In one embodiment, the guide polynucleotide / Cas endonuclease system can be used to insert components of a TET operator repressor / operator / inducer system or components of a sulfonylurea (Su) repressor / operator / inducer system into a plant genome to create or control an inducible expression system.

[0117] In another embodiment, the guide polynucleotide / Cas endonuclease system can be used to allow deletion of a promoter or promoter element, wherein the promoter deletion (or promoter element deletion) results in any one or any combination of the following: permanent inactivation of the locus, increase in promoter activity (increase in promoter strength), increase in promoter tissue specificity, decrease in promoter activity, decrease in promoter tissue specificity, new promoter activity, inducible promoter activity, expansion of the gene expression window, alteration in the timing or developmental progression of gene expression, mutation of DNA binding elements, and / or addition of DNA binding elements. The promoter element to be deleted can be, but is not limited to, a promoter core element, a promoter enhancer element, or a 35S enhancer element. The promoter or promoter fragment to be deleted can be endogenous, artificial, pre-existing, or transgenic to the cell being edited.

[0118] In one embodiment, the nucleotide sequence to be modified can be a terminator, wherein the editing of the terminator includes replacing the terminator (also referred to as "terminator exchange" or "terminator replacement") or the terminator fragment with a different terminator (also referred to as replacement terminator) or terminator fragment (also referred to as replacement terminator fragment), wherein the terminator replacement results in any one or any combination of the following: increased terminator activity, increased terminator tissue specificity, decreased terminator activity, decreased terminator tissue specificity, mutation of DNA binding elements, and / or deletion or addition of DNA binding elements. The terminator (or terminator fragment) to be modified can be an endogenous, artificial, pre-existing, or transgenic terminator (or terminator fragment) for the cell being edited. The replacement terminator (or replacement terminator fragment) can be an endogenous, artificial, pre-existing, or transgenic terminator (or terminator fragment) for the cell being edited.

[0119] The terminator (or terminator element) to be inserted may be endogenous, artificial, pre-existing, or transgenic to the cell being edited.

[0120] In another embodiment, the guide polynucleotide / Cas endonuclease system can be used to allow deletion of terminators or terminator elements, wherein terminator deletion (or terminator element deletion) results in any one or any combination of: increased terminator activity (increased terminator strength), increased terminator tissue specificity, decreased terminator activity, decreased terminator tissue specificity, mutation of DNA binding elements, and / or addition of DNA binding elements. The terminator or terminator fragment to be deleted can be endogenous, artificial, pre-existing, or transgenic to the cell being edited.

[0121] Modifications include 5' caps, 3' poly A tails, riboswitch sequences, stability control sequences, sequences that form dsRNA duplexes, modifications or sequences that target the guide polynucleotide to a subcellular location, modifications or sequences that provide tracking, modifications or sequences that provide protein binding sites, locked nucleic acids (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotides, 2'-fluoro U nucleotides; 2'-O-methyl RNA nucleotides, phosphorothioate bonds, attachment to a cholesterol molecule, attachment to a polyethylene glycol molecule, attachment to a spacer 18 molecule, 5' to 3' covalent attachment, or any combination thereof. These modifications can result in at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of modified or modulated stability, subcellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity to complementary target editing sites, modified resistance to cellular degradation, and increased cellular permeability.

[0122] In some embodiments, the target genomic sequence to be modified is a polyubiquitination site, wherein modification of the polyubiquitination site results in a change in the rate of protein degradation. The ubiquitin tag declares that the protein is to be degraded by the proteasome or autophagy. Known proteasome inhibitors can cause overproduction of proteins. The modification of the DNA sequence encoding the target protein can result in at least one amino acid modification of the target protein, wherein the modification allows polyubiquitination (post-translational modification) of the protein, which results in modification of protein degradation.

[0123] In some embodiments, the target editing site is located in a gene coding region. In some embodiments, the target sequence is located in an intragenic region. In some embodiments, the target sequence is located in a telomere.

[0124] In some embodiments, the methods provided herein result in modification of one or more nucleotides at the target editing site.

[0125] In some embodiments, the modification of the target editing site is a substitution of one or more nucleotides. In some embodiments, the modification of the target editing site is a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0126] In some embodiments, the modification to the target editing site is a deletion of one or more nucleotides. In some embodiments, the modification to the target editing site is a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0127] In some embodiments, the modification to the target editing site is an insertion of one or more nucleotides. In some embodiments, the modification to the target editing site is a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0128] In some embodiments, the target editing site is modified by a donor sequence having one or more insertions, deletions, or substitutions compared to the target editing site. In some embodiments, the target editing site is replaced by a donor sequence.

[0129] By manipulation of the target sequence, the applicant also means epigenetic manipulation of the target editing site. This can be directed to the chromatin state of the target sequence, such as by modification of the methylation state of the target editing site (i.e., addition or removal of methylation or methylation patterns or CpG islands), histone modification, increasing or decreasing the accessibility of the target editing site, or by promoting 3D folding.

[0130] Also provided are methods for probing the function of one or more genes in one or more animal or plant cells, the methods comprising introducing a genetic perturbation using the methods provided herein and determining changes in expression of the one or more genes in the altered cells, thereby probing the function of the one or more genes. In some embodiments, the genetic perturbation is a loss-of-function mutation.

[0131] In some embodiments, the method includes using multiple donor DNAs with different modifications (i.e., insertions, deletions, or substitutions) to the same target. In some embodiments, multiple donor DNAs target promoter regions or coding sequences. In some embodiments, cells with different modifications can then be screened for specific phenotypes. ii. Genetic Engineering of Mammalian Animals

[0132] Also provided herein are methods for gene editing in mammalian cells. In some embodiments, gene editing is directed to a locus involved in a genetic disorder or disease. In some embodiments, the disease or disorder is caused by a mutation in an enzyme. In some embodiments, the genetic disorder is a metabolic disorder.

[0133] Exemplary disorders and genes are amyloid neuropathy (TTR, PALB); amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988); cystic fibrosis (CFTR, ABCC7, CF, MRP7); glycogen storage disease (SLC2A2, GLUT2, G 6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), liver failure, early onset and neurological disorders (SCOD1, SCO1), hepatic lipase deficiency (LIPC), hepatoblastoma, cancer and carcinoma (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN 1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); polycystic kidney and liver disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Other preferred targets include any one or more of the following, including one or more of the following: PCSK9; Hmgcr; SERPINA1; ApoB; LDL; Huntington's disease (huntingtin), hemochromatosis (HEF), Duchenne muscular dystrophy (dystrophin), sickle cell anemia (beta globulin) and Tay-Sachs (hexosaminidase A)

[0134] It should be understood that, in the case of a method of modifying an organism or a mammal or non-human mammal or organism including a human by manipulating a target editing site in a genomic locus of interest, this is applicable to the organism (mammal) as a whole or only to a single cell or cell colony (if the organism is multicellular) from the organism. In the case of humans, for example, the applicant has particularly contemplated single cells or cell colonies and these can preferably be modified ex vivo and then reintroduced. In this case, a biopsy or other tissue or biological fluid sample may be necessary. In this regard, stem cells are also particularly preferred. However, in vivo embodiments are also contemplated.

[0135] The method may be ex vivo or in vitro, for example in cell culture or in an ex vivo or in vitro model (e.g., an organoid or 'animal or plant cell on a chip'). Alternatively, the method may be in vivo, in which case it may also comprise isolating a first cell population from a subject and (re)transplanting a second cell population into the subject. The genetic perturbation may be directed to one or more, or two or more, or three or more, or four or more genes.

[0136] In some embodiments of the invention, knockout models can be generated.

[0137] In some embodiments, delivery is in the form of a vector, which can be a viral vector, such as a lentiviral vector or a baculoviral vector, or preferably an adenovirus / adeno-associated virus vector, but other means of delivery are known and provided (e.g., yeast systems, microvesicles, gene guns / means of connecting the vector to gold nanoparticles). A vector can refer not only to a viral or yeast system (e.g., in which the target nucleic acid can be operably linked to a promoter and controlled by it (in terms of expression, for example, ultimately providing processed RNA)), but also to a nucleic acid delivered directly to a host cell. Although in the methods herein the vector can be a viral vector and this is advantageously AAV, other viral vectors discussed herein, such as lentivirus, can be used. For example, baculovirus can be used for expression in insect cells. These insect cells can in turn be used to produce a large number of other vectors, such as AAV or lentiviral vectors suitable for delivery of the present invention. iii. Genetic engineering of plants

[0138] In certain embodiments, there is provided herein a method for genetically engineering plants. Target polynucleotides / polypeptides include, but are not limited to, herbicide tolerance coding sequences, insecticide coding sequences, nematode coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences that modify plant traits (e.g., yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and / or utilization, fatty acid and oil content and / or composition). More specific target polynucleotides include, but are not limited to, genes that improve crop yield, polypeptides that improve crop desirability, genes encoding proteins that confer resistance to abiotic stress (e.g., drought, nitrogen, temperature, salinity, toxic metals, or trace elements), or those that confer resistance to toxins (e.g., pesticides and herbicides) or biotic stress (e.g., attacks of fungi, viruses, bacteria, insects, and nematodes) and the development of diseases associated with these organisms. General categories of target genes include, for example, genes related to information (e.g., zinc fingers), genes related to communication (e.g., kinases), and genes related to housekeeping (e.g., heat shock proteins). For example, more specific transgenic categories include genes encoding important agronomic traits, insect resistance, disease resistance, herbicide resistance, fertility or sterility, grain characteristics, and commercial products. Genes of interest typically include those involved in oil, starch, carbohydrate, or nutrient metabolism, as well as those affecting kernel size, sucrose loading, etc. These genes can be stacked or combined with other traits.

[0139] In addition to using traditional breeding methods, important agronomic traits such as oil, starch and protein content can also be genetically modified. Modification includes increasing the content of oleic acid, saturated and unsaturated oils, increasing the level of lysine and sulfur, providing the modification of essential amino acids and starch. Protein modification of hordothionin is described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389 (incorporated herein by reference). Another example is a lysine- and / or sulfur-rich seed protein (described in U.S. Patent No. 5,850,016) encoded by soybean 2S albumin and a chymotrypsin inhibitor from barley (described in Williamson, et al., (1987) Eur. J. Biochem [European Journal of Biochemistry] 165: 99-106), the disclosure of which is incorporated herein by reference.

[0140] Commercial traits can also be encoded on the polynucleotide of interest, which can, for example, increase starch for ethanol production or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics, as described, for example, in U.S. Patent No. 5,602,321. Genes such as β-ketothiolase, PHB (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs).

[0141] Derivatives of the coding sequence can be prepared by site-directed mutagenesis to increase the level of a preselected amino acid in the encoded polypeptide. For example, the gene encoding barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application serial number 08 / 740,682, filed November 1, 1996, and WO 98 / 20133, the disclosures of which are incorporated herein by reference. Other proteins include methionine-rich plant proteins, such as protein from sunflower seeds (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, IL), pp. 497-502; incorporated herein by reference); corn (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both incorporated herein by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123, incorporated herein by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0142] Polynucleotides that improve crop yield include dwarfing genes, such as Rht1 and Rht2 (Peng et al. (1999) Nature 400:256-261), and those that promote plant growth, such as ammonium-inducible glutamate dehydrogenase. Polynucleotides that improve crop desirability include, for example, those that provide plants with reduced saturated fat content, those that increase the nutritional value of plants, and those that increase grain protein. Polynucleotides that improve salt tolerance are those that increase or permit plant growth in environments with higher salinity than the native environment of the plant into which one or more salt tolerance genes have been introduced.

[0143] Polynucleotides / polypeptides that affect amino acid biosynthesis include, for example, anthranilate synthase (AS; EC 4.1.3.27), which catalyzes the first reaction in plants, fungi, and bacteria that branches from the aromatic amino acid pathway to tryptophan biosynthesis. In plants, the chemical process of tryptophan biosynthesis is compartmentalized in the chloroplasts. For example, see U.S. Publication 20080050506, incorporated herein by reference. Other target sequences include chorismate pyruvate lyase (CPL), which refers to a gene encoding an enzyme that catalyzes the conversion of chorismate into pyruvate and pHBA. The most fully characterized CPL gene has been isolated from Escherichia coli and has GenBank accession number M96268. See U.S. Patent No. 7,361,811, which is incorporated herein by reference.

[0144] These polynucleotide sequences of interest can encode proteins involved in providing disease or pest resistance. "Disease resistance" or "pest resistance" refers to the avoidance of harmful symptoms in plants as a result of plant-pathogen interactions. Pest resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borer, etc. Disease resistance and insect resistance genes, such as lysozymes or cecropins for protection against bacteria, or proteins such as defensins, glucanases, or chitinases for protection against fungi, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for control of nematodes or insects are all examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as those for fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr) and resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089), etc. Insect resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, and European corn borers. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

[0145] "Herbicide resistance proteins" or proteins expressed by "nucleic acid molecules encoding herbicide resistance" include proteins that confer on cells the ability to tolerate higher concentrations of herbicides than cells that do not express the protein, or tolerate a certain concentration of herbicide for a longer period of time than cells that do not express the protein. Herbicide resistance traits can be introduced into plants by genes encoding resistance to herbicides that inhibit acetolactate synthase (ALS) (particularly sulfonylurea herbicides), genes encoding resistance to herbicides that inhibit glutamine synthase (e.g., phosphinothricin or basta) (e.g., bar genes), genes encoding resistance to glyphosate (e.g., EPSP synthase genes and GAT genes), genes encoding resistance to HPPD inhibitors (e.g., HPPD genes), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and U.S. Provisional Application No. 61 / 401,456, each of which is incorporated herein by reference. The bar gene encodes resistance to the herbicide basast, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and an ALS gene mutant encodes resistance to the herbicide chlorsulfuron.

[0146] Additional selectable markers include genes that confer resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). For example, see Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987) Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; F Dugge et al. (1988) Cell 52:713-22; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-4; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-53; Deuschle et al. (1990) Science 248:480-3; Gossen, (1993) Ph.D. Thesis, University University of Heidelberg, Germany; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-21; Labow et al. (1990) Mol Cell Biol 10:3343-56; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-6; Baim et al. (1991) Proc. Natl. Acad. Sci.USA 88:5072-6; Wyborski et al. (1991) Nucleic Acids Res 19:4647-53; Hillen and Wissman (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al. (1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al. (1988) Biochemistry 27:1094-104; Bonin (1993) Ph.D. Thesis University of Heidelberg [University of Heidelberg, Germany]; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9; Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-4. Commercial traits may also be encoded on one or more genes, which may, for example, increase starch for ethanol production or provide for protein expression. Another important commercial use of transformed plants is the production of polymers and bioplastics, as described, for example, in U.S. Pat. No. 5,602,321. Genes such as β-ketothiolase, PHB (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs).

[0147] Exogenous products include plant enzymes and products as well as products from other sources, including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. Protein levels can be increased, particularly levels of modified proteins with improved amino acid profiles to improve the nutritional value of plants. This is achieved by expressing such proteins with enhanced amino acid content.

[0148] In some embodiments, eukaryotic cells are engineered to produce one or more exogenous proteins in a biosynthetic pathway. In some embodiments, the biosynthetic pathway is used for biofuel production. In some embodiments, the biosynthetic pathway is directed to alcohols. In some embodiments, the biosynthetic pathway is directed to ethanol. In some embodiments, the biosynthetic pathway is used to produce small molecules. In some embodiments, the biosynthetic pathway is used to produce pharmaceuticals. In some embodiments, the biosynthetic pathway is used to produce sterols. In some embodiments, the biosynthetic pathway is directed to hormones. In some embodiments, the biosynthetic pathway is directed to peptide production. In some embodiments, the biosynthetic pathway is directed to terpenes.

[0149] In some embodiments, the eukaryotic cell is engineered so that its progeny no longer replicate. In some embodiments, the eukaryotic cell is a pathogenic cell.

[0150] Transgenes, recombinant DNA molecules, target DNA sequences, and target polynucleotides may comprise one or more DNA sequences for gene silencing. Gene silencing methods involving the expression of DNA sequences in plants are known in the art and include, but are not limited to, cosuppression, antisense inhibition, double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA) interference, intron-containing hairpin RNA (ihpRNA) interference, transcriptional gene silencing, and microRNA (miRNA) interference. iv. Detection

[0151] Those of ordinary skill in the art will appreciate that genetic modification of the target editing site can be detected in a variety of ways. In some embodiments, the method further comprises sequencing the cells. In some embodiments, the method comprises detecting a reporter gene. In some embodiments, the method comprises selecting cells using a selection marker.

[0152] Examples of selectable markers include, but are not limited to, DNA segments comprising restriction enzyme sites; DNA segments encoding products that confer resistance to otherwise toxic compounds (including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT)); DNA segments encoding products that are otherwise absent in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that are readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); generating new primer sites for PCR (e.g., juxtaposition of two DNA sequences that have not been previously juxtaposed), comprising DNA sequences that are inactivated or activated by restriction endonucleases or other DNA modifying enzymes, chemicals, and the like; and comprising DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0153] Additional selectable markers include genes that confer resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). For example, see Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987) Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; F Dugge et al. (1988) Cell 52:713-22; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-4; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-53; Deuschle et al. (1990) Science 248:480-3; Gossen, (1993) Ph.D. Thesis, University University of Heidelberg, Germany; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-21; Labow et al. (1990) Mol Cell Biol 10:3343-56; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-6; Baim et al. (1991) Proc. Natl. Acad. Sci.USA 88:5072-6; Wyborski et al. (1991) Nucleic Acids Res 19:4647-53; Hillen and Wissman (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al. (1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al. (1988) Biochemistry 27:1094-104; Bonin (1993) Ph.D. Thesis University of Heidelberg [University of Heidelberg, Germany]; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother [Antimicrob Agents and Chemotherapeutics] 36:913-9; Hlavka et al., (1985) Handbook of Experimental Pharmacology [Handbook of Experimental Pharmacology], Vol. 78 (Springer-Verlag, Berlin); Gill et al., (1988) Nature [Nature] 334:721-4. C. Nucleic acid

[0154] On the one hand, the present disclosure provides nucleic acids encoding HDR promoters. In some embodiments, a composition is provided herein comprising nucleic acids encoding one or more of the following: i) at least one sequence-specific nuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) a nuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, the nucleic acid is located in one or more vectors. In some embodiments, the nucleic acid is in a vector.

[0155] In some embodiments, the nucleic acid encodes at least one sequence-specific endonuclease. In some embodiments, the nucleic acid comprises a donor template DNA molecule having homology to the target editing site. In some embodiments, the nucleic acid encodes an HDR promoter. In some embodiments, the nucleic acid encodes a single-stranded DNA annealing protein (SSAP). In some embodiments, the nucleic acid encodes an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product. In some embodiments, the nucleic acid encodes a single-stranded DNA binding protein (SSB). In some embodiments, the nucleic acid is an expression construct or vector. In some embodiments, the expression construct or vector comprises a nucleic acid.

[0156] In certain embodiments, nucleic acid encodes gene editing molecules.In certain embodiments, nucleic acid encodes sequence-specific endonucleases.In certain embodiments, the nucleic acid encoding sequence-specific endonucleases includes the polynucleotides and guide RNA of the nuclease guided by RNA or the nuclease encoding RNA or the polynucleotides encoding guide RNA.In certain embodiments, nucleic acid encoding RNA guides DNA endonucleases, II type Cas nucleases, Cas9 nucleases, V type Cas nucleases, Cas12a nucleases, Cas12b nucleases, Cas12c nucleases, CasY nucleases, CasX nucleases or engineered nucleases.In certain embodiments, nucleic acid encodes zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL-effector nucleases), Argonautes, meganucleases or engineered meganucleases.In certain embodiments, nucleic acid encodes one or more sequence-specific endonucleases or sequence-specific endonucleases and guide RNA that cut single DNA strands at two different DNA sequences at the target editing site. In some embodiments, the nucleic acid encodes a sequence-specific endonuclease comprising at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof. In some embodiments, the nucleic acid encodes a sequence-specific endonuclease comprising Cas9 and / or Cas12a, and the guide RNA molecule has at least one base mismatch with the DNA sequence in the target editing site.

[0157] In some embodiments, the nucleic acid comprises a donor DNA molecule. In some embodiments, the nucleic acid comprises a donor template DNA. In some embodiments, the donor DNA molecule is provided on a circular DNA vector, a Geminivirus replicon, or as a linear DNA fragment. In some embodiments, the donor DNA molecule is flanked by endonuclease recognition sequences.

[0158] In some embodiments, the donor DNA molecule comprises a modified sequence of a genomic DNA target editing site. In some embodiments, the donor DNA molecule comprises a substitution of one or more nucleotides compared to the target editing site. In some embodiments, the donor DNA molecule comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0159] In some embodiments, the donor DNA molecule comprises a deletion of one or more nucleotides compared to the genomic target editing site. In some embodiments, the donor DNA molecule comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0160] In some embodiments, the donor DNA molecule comprises an insertion of one or more nucleotides compared to the genomic target editing site. In some embodiments, the insertion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0161] In some embodiments, the nucleic acid encoding the sequence-specific endonuclease comprises an RNA-guided nuclease, and the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and adjacent to the pre-spacer adjacent motif (PAM) sequence. In some embodiments, the nucleic acid encodes a sequence-specific endonuclease that provides a 5' overhang at the target editing site after cutting. In some embodiments, the nucleic acid encodes an SSAP that provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules. In some embodiments, the nucleic acid encodes an SSAP comprising a RecT / Redβ family protein, an ERF family protein, or a RAD52 family protein. In some embodiments, the nucleic acid encodes a RecT / Redβ family protein comprising a Rac bacterial prophage RecT protein, a phage λβ protein, a phage SPP1 35 protein, a related protein with equivalent SSAP activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, 2 or 3. In some embodiments, the nucleic acid encodes an ERF family protein comprising 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 to SEQ ID NO: 4. In some embodiments, the nucleic acid encodes a RAD52 family 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 to SEQ ID NO: 5, 6, or 7.

[0162] In some embodiments, the nucleic acid encodes an exonuclease. In some embodiments, the nucleic acid encodes an exonuclease, wherein a linear dsDNA molecule is a preferred substrate for the exonuclease. In some embodiments, a linear dsDNA molecule comprising a phosphorylated 5' end is a preferred substrate for the exonuclease. In some embodiments, the exonuclease has a 5' to 3' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. In some embodiments, the exonuclease has a 3' to 5' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. In some embodiments, the exonuclease comprises a bacteriophage lambda exo protein, a Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpes virus SOX protein, a UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, an exonuclease III, a Trex2 exonuclease, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144 or 145. In some embodiments, the exonuclease comprises T7 phage exonuclease, Escherichia coli exonuclease III, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 143 or 144.

[0163] In some embodiments, the nucleic acid encodes a single-stranded DNA binding protein (SSB). In some embodiments, the nucleic acid encodes an SSB and an SSAP. In some embodiments, the nucleic acid encodes a single-stranded DNA binding protein (SSB) and an SSAP obtained from the same host organism. In some embodiments, the single-stranded DNA binding protein (SSB) is a bacterial SSB or optionally an Enterobacteriaceae species SSB. In some embodiments, the SSB is an Escherichia species, a Shigella species, an Enterobacter species, a Klebsiella species, a Serratia species, a Pantoea species or a Yersinia species SSB. In some embodiments, the SSB comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 31, 34-131 or 132.

[0164] In some embodiments, the nucleic acid encodes an SSAP, exonuclease, and / or SSB protein further comprising an operably linked nuclear localization signal (NLS) and / or cell penetrating peptide (CPP). In some embodiments, the nucleic acid encodes a protein for expression in a plant cell. In some embodiments, the SSAP, exonuclease, and / or single-stranded DNA binding protein further comprises an operably linked 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.

[0165] In some embodiments, the nucleic acids provided herein encoding i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB) are each operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a plant-specific promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a 35S promoter. In some embodiments, the promoter is a ubiquitin promoter. In some embodiments, the promoter is an actin promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is an EF1a promoter. In some embodiments, the promoter is a human ACTB promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a T7 promoter. In some embodiments, the site-specific nuclease and / or its guide RNA for the CRISPR / Cas-based nuclease is expressed under the control of an inducible promoter. In this configuration, the initiation of the genome editing process can be induced when the concentrations of the other components of the system are not rate-limiting.

[0166] In some embodiments, the nucleic acids provided herein are provided in one or more vectors. In some embodiments, the nucleic acids provided herein are provided in one vector. In some embodiments, the nucleic acids provided herein are provided in two vectors. In some embodiments, the nucleic acids provided herein are provided in three vectors. In some embodiments, the nucleic acids provided herein are provided in four vectors. In some embodiments, the nucleic acids provided herein are provided in five vectors.

[0167] In some embodiments, provided herein are vectors encoding i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, provided herein are vectors encoding HDR-promoting elements. In some embodiments, provided herein are vectors encoding: a single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB). In some embodiments, provided herein are vectors encoding at least one sequence-specific endonuclease and a donor template.

[0168] Also provided herein is a first vector comprising a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB), and a second vector comprising a donor template DNA and a guide RNA.

[0169] In some embodiments, the nucleic acid is optimized for expression in a specific cell type. In some embodiments, the nucleic acid is optimized for expression in a specific species. In some embodiments, the nucleic acid is optimized for expression in plant cells. In some embodiments, the nucleic acid is optimized for expression in mammalian cells. In some embodiments, the nucleic acid comprises a protein coding sequence, such as an exonuclease, an SSB protein, and / or an SSAP. In some embodiments, the protein coding sequence is codon-optimized for translation in plant cells. In some embodiments, the protein coding sequence is codon-optimized for translation in mammalian cells.

[0170] In certain embodiments, the length of the donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800 or 1000 base pairs. For example, the length of the donor DNA template homology arm can be about 20 to about 1000, about 50 to about 1000, about 100 to about 1000, about 200 to about 1000, or about 600 to about 1000 base pairs. In some embodiments, the length of the donor DNA template homology arm is between about 400 to about 800 base pairs. In some embodiments, the length of the donor DNA template homology arm is less than 250 base pairs. In some embodiments, the length of the donor DNA template homology arm is less than 100 base pairs.

[0171] In certain embodiments, the GC content of the donor DNA template homology arms is altered. In certain embodiments, the GC content is maximized.

[0172] In some embodiments, the nucleic acids provided herein are modified for expression in a certain cell type. In some embodiments, the nucleic acids provided herein are modified for expression in eukaryotic cells. In some embodiments, the nucleic acids are modified for expression in plant or animal cells. In some embodiments, the nucleic acids are modified for mammalian cells. In some embodiments, the nucleic acids are modified for mouse or primate cells. In some embodiments, the nucleic acids are modified for human cells. In some embodiments, the nucleic acids are modified for mouse cells.

[0173] Methods for modifying nucleic acid compositions expressed in specific cell types are well known in the art. In certain embodiments, the GC (guanine-cytosine) content of the nucleotides provided herein is changed. In certain embodiments, the nucleic acid provided herein is codon optimized for specific cell types such as eukaryotic cells. i. Viral vectors

[0174] In one aspect, the present disclosure provides vectors comprising any of the nucleic acids disclosed herein for expression in mammalian cells. In some embodiments, the vector comprises an expression construct. In some embodiments, the vector comprises a nucleic acid encoding an HDR facilitator (e.g., SSAP, exonuclease, and / or SSB protein), a sequence-specific endonuclease, and / or a donor template DNA molecule.

[0175] In some embodiments, provided herein is a vector comprising a nucleic acid encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and / or v) a single-stranded DNA binding protein (SSB).

[0176] In some embodiments, the first vector encodes one or more of the following: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to the target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, the second vector encodes one or more of the following: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to the target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, the first vector does not encode at least one of the sequence-specific endonuclease, the donor template DNA molecule, the SSAP, the exonuclease, and the SSB protein. In some embodiments, at least one of the sequence-specific endonuclease, donor template DNA molecule, SSAP, exonuclease, and SSB protein not encoded by the first vector is encoded by the second vector. In some embodiments, the components are encoded by the first and second vectors as shown in Table B below. Table B: Combinations of components encoded by the first and second vectors

[0177] In some embodiments, the sequence-specific endonuclease, the donor template DNA molecule, the SSAP, the exonuclease, and the SSB are provided in various combinations in three vectors, for example, a first vector comprising the sequence-specific endonuclease, a second vector comprising the donor template DNA, and a third vector comprising the SSAP, the exonuclease, and the SSB, or a first vector comprising the sequence-specific endonuclease, the donor template DNA, and the SSAP, a second vector comprising the exonuclease, and a third vector comprising the SSB.

[0178] In some embodiments, the sequence-specific endonuclease, the donor template DNA molecule, the SSAP, the exonuclease, and the SSB are provided in various combinations in four vectors, such as a first vector comprising the sequence-specific endonuclease, a second vector comprising the donor template DNA, a third vector comprising the SSAP, and a fourth vector comprising the exonuclease and the SSB, or a first vector comprising the sequence-specific endonuclease and the donor template DNA, a second vector comprising the SSAP, a third vector comprising the exonuclease, and a fourth vector comprising the SSB.

[0179] In some embodiments, the sequence-specific endonuclease, donor template DNA molecule, SSAP, exonuclease, and SSB are provided in five vectors.

[0180] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a parvoviral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a herpes virus vector. In some embodiments, the vector is a baculoviral vector.

[0181] In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5. In some embodiments, the vector is a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD 114, or a variant thereof. In some embodiments, the vector is a rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0182] In some embodiments of the above methods, the vector is an rAAV vector. In some embodiments, the expression construct encoding an HDR promoter (e.g., SSAP, exonuclease and / or SSB protein), a sequence-specific endonuclease and / or a donor template DNA molecule is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the expression construct is flanked by two AAV ITRs. In some embodiments, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, cattle AAV or mouse AAV serotype ITRs. In some embodiments, AAV ITRs are AAV2 ITRs. In some embodiments, the vector further comprises a filler nucleic acid. In some embodiments, the filler nucleic acid is located between the promoter and the nucleic acid encoding the expression construct. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid and a second nucleic acid, the first nucleic acid encoding an HDR facilitator (e.g., SSAP, exonuclease and / or SSB protein), a sequence-specific endonuclease and / or a donor template DNA molecule, and the second nucleic acid encoding an HDR facilitator (e.g., SSAP, exonuclease and / or SSB protein), a sequence-specific endonuclease and / or a donor template DNA molecule. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion in the D region and comprises a mutation in the terminal resolution sequence. In some embodiments, the present invention provides cells comprising any vector described herein (e.g., an rAAV vector).

[0183] In some embodiments of the above methods, the vector encoding the HDR promoter (e.g., SSAP, exonuclease and / or SSB), sequence-specific endonuclease and / or donor template DNA molecule is in a viral particle, wherein the viral particle is an AAV particle encapsulating a rAAV vector, an adenoviral particle encapsulating a recombinant adenoviral vector, a lentiviral particle encapsulating a recombinant lentiviral vector, or an HSV particle encapsulating a recombinant HSV vector. In some embodiments, the viral particle is an adenoviral particle encapsulating a recombinant adenoviral vector. In some embodiments, the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3. In some embodiments, the adenoviral particle comprises an adenovirus serotype 2 capsid, or a variant of an adenovirus serotype S capsid. In some embodiments, the viral particle is a lentiviral particle that encapsulates a recombinant lentiviral vector. In some embodiments, the lentiviral particle comprises a capsid pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a variant thereof. In some embodiments, the viral particle is an HSV particle. In some embodiments, the HSV particle is a rHSV-1 particle or a rHSV-2 particle.

[0184] In some embodiments of the above methods, the present invention provides recombinant AAV particles comprising any of the rAAV vectors described herein. In some embodiments, the AAV viral particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAVDJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITRs and the capsid of the rAAV viral particles are derived from the same AAV serotype. In some embodiments, the ITRs and the capsid of the rAAV viral particles are derived from different AAV serotypes. In some embodiments, the ITR is derived from AAV2 and the capsid of the rAAV particle is derived from AAV1. The present invention provides a vector comprising the expression construct of any one of the embodiments described herein. In some embodiments, the expression construct encodes an HDR promoter (e.g., SSAP, exonuclease and / or SSB), a sequence-specific endonuclease and / or a donor template DNA molecule. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentiviral vector or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, or a variant of adenovirus serotype S. In some embodiments, the vector is a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a variant thereof. In some embodiments, the vector is a rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0185] In some embodiments, the vector comprises a selectable marker.

[0186] In some embodiments of the above methods, the viral particles are in a composition (eg, a pharmaceutical composition). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. ii. Other carriers

[0187] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a plant transformation vector. In some embodiments, the vector is a vector for Agrobacterium-mediated transient expression or stable transformation in tissue culture or plant tissue.

[0188] Exemplary systems using recombinant plasmid vectors compatible with the present invention include, but are not limited to, "cointegration" and "binary" systems. In the "cointegration" system, a shuttle vector containing the gene of interest is inserted into a non-oncogenic plasmid by genetic recombination, which contains the cis- and trans-acting elements required for plant cell transformation, for example, in the pMLJ1 shuttle vector and the non-oncogenic plasmid pGV3850. The second system is called the "binary" system, in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. Other necessary functions are provided by non-oncogenic plasmids, such as the pBIN19 shuttle vector and the non-oncogenic plasmid PAL4404. These and other vectors that can be used in these systems are commercially available. D.cells

[0189] On the one hand, the present disclosure provides eukaryotic cells comprising HDR promoters. In some embodiments, the eukaryotic cells comprise genome editing molecules and HDR promoters. In some embodiments, the cells are host cells. In some embodiments, the cells are cells modified according to this method. In some embodiments, the genome editing molecules comprise (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cutting the DNA sequence at the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site. In some embodiments, the HDR promoter comprises a single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB).

[0190] On the other hand, the present disclosure provides eukaryotic cells produced by the methods provided herein. In some embodiments, the modification of the target editing site of the eukaryotic cell genome includes providing a genome editing molecule and an HDR promoter to the eukaryotic cell, wherein the genome editing molecule comprises (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cuts the DNA sequence at the target editing site and (ii) a donor template DNA molecule with homology to the target editing site; and wherein the HDR promoter comprises SSAP, an exonuclease and an SSB protein that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product. In some embodiments, the cell has a genomic feature produced by modification according to this method. In some embodiments, the nuclease cleavage site is removed. In some embodiments, nucleic acid sequence tags are beneficial.

[0191] In some embodiments, provided herein are host cells comprising one or more vectors comprising i) a nucleic acid encoding at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a nucleic acid encoding a single-stranded DNA annealing protein (SSAP), iv) a nucleic acid encoding an exonuclease that at least partially converts a double-stranded DNA substrate into a single-stranded DNA product, and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB). In some embodiments, the host cell comprises a vector encoding i) a nucleic acid encoding at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a nucleic acid encoding a single-stranded DNA annealing protein (SSAP), iv) a nucleic acid encoding an exonuclease that at least partially converts a double-stranded DNA substrate into a single-stranded DNA product, and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB). In some embodiments, the cell comprises a first vector comprising i) a nucleic acid encoding at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, and a second vector comprising iii) a nucleic acid encoding a single-stranded DNA annealing protein (SSAP), iv) a nucleic acid encoding an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB).

[0192] Furthermore, the methods of the present disclosure can be used to increase HDR-mediated genome modification in eukaryotic cells, to produce eukaryotic cells with genome modifications, and / or to genetically engineer eukaryotic cells as described herein.

[0193] In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture. In some embodiments, the cell is isolated. In some embodiments, the cell is obtained from a living organism and maintained in cell culture. In some embodiments, the cell is a unicellular organism. In some embodiments, the cell is inside an organism. In some embodiments, the cell is an organism. In some embodiments, the cell is a unicellular eukaryotic cell, a protozoan cell, a cell from a plant, an algae cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum sparganum, patens), C. agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., ungulates (e.g., pigs, cows, goats, sheep); rodents (e.g., rats, mice); non-human primates; humans; felines (e.g., cats); canines (e.g., dogs); etc.), etc. In some embodiments, the cell is a cell that is not derived from a natural organism (e.g., the cell can be a synthetically manufactured cell; also referred to as an artificial cell). In some embodiments, the cell is in a cell culture (e.g., an in vitro cell culture). In some embodiments, the cell is one of a collection of cells. In some embodiments, the cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, the cell is a plant cell or is derived from a plant cell. In some embodiments, the cell is an animal cell or is derived from an animal cell. In some embodiments, the cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, the cell is a rodent cell or is derived from a rodent cell. In some embodiments, the cell is a human cell or is derived from a human cell. In some embodiments, the cell is a non-human animal cell or is derived from a non-human animal cell. In some embodiments, the cell is a non-human mammalian cell or is derived from a non-human mammalian cell. In some embodiments, the cell is a fungal cell or is derived from a fungal cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is an arthropod cell. In some embodiments, the cell is a protozoan cell. In some embodiments, the cell is a worm cell. In some embodiments, the cell is a non-mammalian cell.In some embodiments, the cell is a fish cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a fruit fly cell. In some embodiments, the cell is a Drosophila melanogaster cell. In some embodiments, the cell is a nematode cell. In some embodiments, the cell is a Caenorhabditis elegans cell. In some embodiments, the cell is a roundworm cell.

[0194] In some embodiments, the cell is a progenitor cell comprising one or more of: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that is capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB), wherein the progenitor cell does not comprise at least one of i)-v), and wherein at least one of i)-v) not comprised by the progenitor cell is subsequently provided by delivering a polypeptide, DNA, or mRNA to the progenitor cell and / or sexually hybridizing the progenitor cell. For example, in some embodiments, the progenitor cell lacks one or more components of i)-v) and is transformed with the missing components. i. Plant cells

[0195] In some embodiments, the eukaryotic cell is a plant cell. In some embodiments, the eukaryotic cell comprising an HDR promoter is a plant cell. In addition, the methods of the present disclosure can be used to increase HDR-mediated genome modification in plant cells, manufacture plant cells with genome modification, and / or genetically engineer plant cells. In some embodiments, the methods disclosed herein include editing plant cells. In some embodiments, the methods disclosed herein include performing genome modification in plant cells. In some embodiments, the methods disclosed herein include modifying a target locus in the genome of a plant cell. In some embodiments, the methods disclosed herein include increasing HDR-mediated genome modification in plant cells.

[0196] In certain embodiments, cell is the plant cell or plant protoplast that is separated (that is, not being positioned at unseparated or complete plant tissue, plant part or whole plant).In certain embodiments, plant cell is obtained from any plant part or tissue or callus.In certain embodiments, culture comprises the plant cell obtained from following: plant tissue, cultivated plant tissue explant, whole plant, complete node bud, stem tip or stem tip meristem, root tip or root tip meristem, lateral tissue, intercalary meristem, seedling, whole seed, half seed or other seed fragments, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, callus or plant cell suspension.In certain embodiments, plant cell is derived from the immature embryo of monocotyledonous plant (for example maize, wheat, Chinese sorghum or rice) or the L1 or L2 layer of mature embryo.

[0197] In certain embodiments, vegetable cell is positioned at not separated or complete plant tissue, plant part, plant explant or whole plant.In certain embodiments, vegetable cell can be positioned at following: complete node bud, the plant tissue explant of cultivation, stem tip or stem tip meristem, root tip or root tip meristem, lateral tissue, intercalary meristem, seedling, whole seed, half seed or other seed fragments, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root or callus.In certain embodiments, employed explant comprises immature embryo.Immature embryo (for example immature corn embryo) comprises 1.8-2.2mm embryo, 1-7mm embryo and 3-7mm embryo.In certain embodiments, above-mentioned embryo is obtained from seed, leaf base, leaf, leaf tip, immature inflorescence, tassel, immature female spike and filament in mature female spike source. In various aspects, the explant for conversion of plant origin comprises immature embryo, 1.8-2.2mm embryo, 1-7mm embryo and 3.5-7mm embryo.On the one hand, embryo can be derived from the seed, leaf base, leaf from mature plant, leaf tip, immature inflorescence, tassel, immature female spike and filament in mature female spike source.In certain embodiments, plant cell is pluripotent plant cell (for example, stem cell or meristematic cell).In certain embodiments, plant cell is positioned at the immature embryo of monocotyledon (for example maize, wheat, sorghum or rice) or the L1 or L2 layer of mature embryo.

[0198] In certain embodiments, the plant cell is a haploid, diploid, or polyploid plant cell or plant protoplast, such as those obtained from a haploid, diploid, or polyploid plant, plant part or tissue, or callus tissue. In certain embodiments, the plant cells in culture (or regenerated plants, progeny seeds, and progeny plants) are haploid or can be induced to become haploid; techniques for making and using haploid plants and plant cells are known in the art, see, for example, methods for generating haploids in Arabidopsis thaliana by crossing a wild-type line with a haploid-inducing line expressing an altered form of the centromere-specific histone CENH3, as described in Maruthachalam and Chan in “How to make haploid Arabidopsis thaliana,” a protocol available at www[dot]openwetware[dot]org / images / d / d3 / Haploid_Arabidopsis_protocol[dot]pdf; (Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038 / ncomms6334). Haploids can also be obtained in a wide variety of monocots (e.g., maize, wheat, rice, sorghum, barley) or dicots (e.g., soybean, Brassica species, including canola, cotton, tomato) by crossing plants containing a mutated CENH3 gene with wild-type diploid plants to produce haploid progeny, as disclosed in U.S. Pat. No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid-induced maize lines that can be used to obtain haploid maize plants and / or cells include Stock 6, MHI (Moldovian Haploid Inducer), indeterminate gametophyte (ig) mutations, KEMS, RWK, ZEM, ZMS, KMS, and the transgenic haploid inducer lines disclosed in U.S. Pat. No. 9,677,082, which is incorporated herein by reference in its entirety. Examples of haploid cells include, but are not limited to, plant cells obtained from haploid plants and plant cells obtained from reproductive tissue, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, megagametophytes, and microspores.In certain embodiments where the plant cell or plant protoplast is haploid, the genetic complement can be doubled to produce a doubled haploid plant cell or plant protoplast, wherein the complement of the gene or allele is homozygous, by performing chromosome doubling (e.g., spontaneous chromosome doubling by meiosis without reduction, or by using a chromosome doubling agent, such as colchicine, oryzalin, trifluralin, natron, nitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in the plant cell or plant protoplast, wherein the complement of the gene or allele is homozygous; yet other embodiments include regenerating a double haploid plant from a double haploid plant cell or plant protoplast. Another embodiment relates to a hybrid plant having at least one parent plant, the parent plant being a double haploid plant provided by the method. The generation of double haploid plants provides homozygosity within one generation, without the need for several generations of self-pollination to obtain homozygous plants. In any case where it is desired to establish genetic purity (i.e., homozygosity) in the shortest possible time, it is advantageous to use double haploids. Doubled haploid production is particularly advantageous in slow-growing plants, such as fruit and other trees, or for producing hybrid plants that are the offspring of at least one doubled haploid plant.

[0199] In certain embodiments, the plant cell is obtained from or is located in any monocot or dicot species of interest, such as field crop plants, fruit plants and trees, vegetables, trees, and ornamental plants, including ornamental flowers, shrubs, trees, ground cover plants, and turf grasses. In certain non-limiting embodiments, the plant material is obtained from or is located in alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cocoa (Theobroma cacao), canola and rapeseed or rapeseed (Brassicanapus), carnation (Dianthus caryophyllus), carrot (Daucus carota sativus), cassava (Manihot esculentum), cherries (Prunus avium), chickpeas (Cider arietinum), endive (Cichorium intybus), red and other peppers of the genus Capsicum (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemums (Chrysanthemum spp.), coconuts (Cocos nucifera), coffee (Coffea spp.), including Coffea arabica (Coffea spp.). arabica and Coffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), blackcurrant and gooseberry (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linumusitatissumum L.), geranium (Pelargonium spp.), grapefruit (Citrusx paradisi), grapevine (Vitus spp.) (including wine grape (Vitus vinifera)), guava (Psidium guajava), hemp, hops (Humulus lupulus), iris (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), lime (Citrus spp.), corn (Zea mays), mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis melo), millet (Setaria spp.), barnyard grass (Echinochloa spp.), (Eleusine spp.), Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), olives (Oleaeuropaea), onions (Allium cepa), oranges (Citrus sinensis), papayas (Carica papaya), peaches and nectarines (Prunus persica), pears (Pyrus spp.), peas (Pisa sativum), peanuts (Arachis hypogaea), peonies (Paeonia spp.), morning glories (Petunia spp.), pineapples (Ananascomosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa), poplar (Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbita pepo), rice (Oryza sativa L.), rose (Rosa spp.), rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L.), sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycine max L.), squash (Cucurbita pepo), pepo), strawberries (Fragaria spp., Fragaria xananassa), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus annus), sweet potato (Ipomoea batatas), orange (Citrustangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum), tulip (Tulipa spp.), radish (Brassica rapa rapa), walnut (Juglans spp. L.), watermelon (Citrulus lanatus), wheat (Tritium aestivum), or yam (Discorea spp.). ii. Mammalian cells

[0200] In some embodiments, the eukaryotic cell comprising an HDR promoter is an animal cell. In some embodiments, the animal cell is a mammalian cell. In addition, the method of the present disclosure can be used to increase the HDR-mediated genome modification in the animal cell, manufacture animal cells with genome modification, and / or genetically engineer the animal cell. In some embodiments, these methods can be used to increase the HDR-mediated genome modification, manufacture cells with genome modification and / or genetically engineer the mammalian cell. In some embodiments, the method disclosed herein includes editing animal cells, for example, mammalian cells. In some embodiments, the method disclosed herein includes performing genome modification in animal cells (for example, mammalian cells). In some embodiments, the method disclosed herein includes modifying the target locus in the animal cell, for example, mammalian cells. In some embodiments, the method disclosed herein includes increasing the HDR-mediated genome modification in the animal cell (for example, mammalian cells).

[0201] In some embodiments, the cell is an animal cell from any multicellular vertebrate or invertebrate. In some embodiments, the animal is a model organism for biological, physiological or genetic research. Therefore, in some embodiments, the animal is selected from: mouse (Mus musculus), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), cat (Felis sylvestris catus), chicken (Gallus gallus), dog (Canis lupus familiaris), guinea pig (Caviaporcellus), rat (Rattus norvegicus) and nematode (Caenorhabditis elegans). In some embodiments, the animal is a domesticated or cultured animal.Thus, in some embodiments, the animal is selected from the group consisting of goats (Capra aegagrus hircus), pigs (Sus scrofadomesticus), sheep (Ovis aries), cattle (Bos taurus), cats (Felis catus), donkeys (Equus africanus asinus), ducks (Anas platyrhynchos domesticus), buffaloes (including Bubalus bubalisbubalis and Bubalus bubalis carabenesis), honey bees (Apis mellifera), including the Italian subspecies (A. mellifera ligustica), European black honey bees (A. mellifera mellifera), Carniola honey bees (A. mellifera carnica), Caucasian honey bees (A. mellifera caucasia), and Greek honey bees (A. mellifera macedoniana). cecropia), dromedary camel (Camelus dromedarius), horse (Equus ferns caballus), silkworm (Bombyx mori), pigeon (Columba livia), goose (Anser domesticus and Ansercygnoides domesticus), yak (Bos grunniens), Bactrian camel (Camelus bactrianus), llama (Lama glama), alpaca (Vicugna pacos), guinea fowl (Numida meleagris), ferret (Mustela putorius furo), turkey (Meleagris striata), and rooster (Meleagris truncatus). gallopavo, grass carp, silver carp, common carp, Nile tilapia, bighead carp, catla (Indian carp), crucian carp, Atlantic salmon, rohu (roho labeo), milkfish, rainbow trout, Wuchang bream, snakehead carp, northern snakehead and spotted catfish.

[0202] In certain embodiments, the cell is derived from a cell line, for example, a mammalian cell line or a human cell line. A variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, A549, HEK-293, 293T, MF7, K562, Caco-2, HeLa cells and transgenic varieties thereof. In certain embodiments, the cell is an HEK-293 cell. In certain embodiments, the cell is a Chinese hamster ovary (CHO) cell. Cell lines can be obtained from various sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassus, Virginia)). In certain embodiments, cells transfected with one or more nucleic acids as described herein (e.g., vectors encoding HDR promoters) are used to establish a new cell line comprising one or more vector-derived sequences to establish a new cell line comprising a modification to a target nucleic acid.

[0203] In some embodiments, the cell is a primary cell, for example, a mammalian primary cell or a human primary cell. For example, the culture of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. In some embodiments, primary cells are harvested from an individual by any known method. For example, leukocytes can be harvested by apheresis, leukocyte apheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Suitable solutions can be used to disperse or suspend the harvested cells. Such solutions can generally be balanced salt solutions (e.g., physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors, combined with an acceptable low concentration buffer. The buffer can include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately or stored (e.g., by freezing). Frozen cells can be thawed and can be reused. Cells can be frozen in DMSO, serum, culture buffer (eg, 10% DMSO, 50% serum, 40% buffered culture medium), and / or some other such solution commonly used to preserve cells at freezing temperatures.

[0204] In some embodiments, the cell is a human cell. In some embodiments, the cell is a germline cell. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a post-mitotic cell. In some embodiments, the cell is an immune cell, such as a T cell, a natural killer (NK) cell, or a macrophage. In some embodiments, the cell is a human T cell obtained from a patient or a donor. The methods provided herein can be used to modify the target nucleic acid in primary T cells for use in immunotherapy. In some embodiments, the methods provided herein are used to produce CAR-T cells, for example, by editing the genome of the T cell to introduce an expression construct expressing a chimeric antigen receptor (CAR). In some embodiments, the methods provided herein are used to modify immune cells ex vivo. In some embodiments, the methods provided herein are used to produce CAR-T cells ex vivo. In some embodiments, the methods disclosed herein include editing human cells. In some embodiments, the methods disclosed herein include performing genome modification in human cells. In some embodiments, the methods disclosed herein include modifying the target locus in human cells. In some embodiments, the methods disclosed herein include increasing HDR-mediated genome modification in human cells.

[0205] In some embodiments, the cell is a stem cell or progenitor cell. In some embodiments, the cell is an undifferentiated cell. In some embodiments, the cell is a human stem cell or progenitor cell. In some embodiments, the cell is a mammalian stem cell or progenitor cell. In some embodiments, the cell is an adult stem cell, an embryonic stem cell, an induced pluripotent (iPS) cell or a progenitor cell (e.g., a cardiac progenitor cell, a neural progenitor cell, etc.). In some embodiments, the cell is a hematopoietic stem cell (HSC). In some embodiments, the cell is a mesenchymal stem cell (MSC). In some embodiments, the cell is a neural stem cell. In some embodiments, the cell is an epithelial stem cell. The cell may include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.

[0206] In some embodiments, the cell is a diseased cell, e.g., a diseased mammalian cell or a diseased human cell. The diseased cell may have altered metabolic, gene expression, and / or morphological characteristics. In some embodiments, the cell has a genome with a genetic variation associated with the disease. In some embodiments, the cell has a SNP associated with the disease. In some embodiments, the cell's genome has a genetic marker associated with the disease. In some embodiments, the cell has a deleterious mutation. In some embodiments, the cell has a disease-causing mutation. In some embodiments, the cell has a mutant allele associated with the disease. In some embodiments, the cell has a loss-of-function mutation. In some embodiments, the cell has a disease genotype. In some embodiments, the cell has a disease phenotype. In some embodiments, the cell has a genetic defect. In some embodiments, the cell has an oncogenic mutation. In some embodiments, the cell has an integrated and / or stably maintained virus. In some embodiments, a retrovirus is integrated into the cell's genome. In some embodiments, a lentivirus is integrated into the cell's genome. In some embodiments, the cell has a persistent viral infection. In some embodiments, the cell has HIV. In some embodiments, the cell has an integrated copy of the HIV genome. In some embodiments, the cell is virally infected. In some embodiments, the cell has a latent viral infection. In some embodiments, the cell is infected with a herpes virus. In some embodiments, the cell is infected with human herpesvirus 6 or 7. In some embodiments, the cell is infected with herpes simplex virus type 1 or type 2. In some embodiments, the cell is infected with varicella-zoster virus. In some embodiments, the cell is infected with human papillomavirus. In some embodiments, the cell is infected with Epstein-Barr virus. The diseased cell can be a cancer cell, a diabetic cell, or an apoptotic cell. The diseased cell can be a cell from a patient. Exemplary diseases can include genetic disorders, infectious diseases, blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, etc. In some embodiments, the cell is derived from a patient. In some embodiments, the cell is modified in vitro. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is an embryonic cell. In some embodiments, the cell is an embryonic stem cell.

[0207] In some embodiments, the methods provided herein are used to genetically modify diseased cells, for example, diseased mammalian cells or diseased human cells. In some embodiments, the methods provided herein are used to genetically modify diseased cells. In some embodiments, the methods provided herein are used to insert the wild-type allele of a gene into a diseased cell. In some embodiments, the methods provided herein are used to correct harmful mutations in diseased cells. In some embodiments, the methods provided herein are used to genetically modify oncogenes. In some embodiments, the methods provided herein are used to genetically modify the alleles of genes associated with a disease. In some embodiments, the methods provided herein are used to insert the healthy alleles of a gene. In some embodiments, the methods provided herein are used to insert the alleles of a gene that is not related to the disease. In some embodiments, the methods provided herein are used to remove integrated or stably maintained viruses, such as slow viruses, retroviruses, or herpes viruses, from the cell genome. iii. Fungal cells

[0208] In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell comprising an HDR promoter is a fungal cell. In addition, the methods of the present disclosure can be used to increase HDR-mediated genome modification in fungal cells, manufacture fungal cells with genome modification, and / or genetically engineer fungal cells. In some embodiments, the methods disclosed herein include editing fungal cells. In some embodiments, the methods disclosed herein include performing genome modification in fungal cells. In some embodiments, the methods disclosed herein include modifying a target locus in a fungal cell. In some embodiments, the methods disclosed herein include increasing HDR-mediated genome modification in a fungal cell.

[0209] In some embodiments, the fungal cell is a cell derived from a multicellular fungus. In some embodiments, the cell is an Ascomycete cell. In some embodiments, the cell is a unicellular fungus. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a fungal cell of the following genus: Aspergillus, Candida, Cochliobolus, Echinococcus, Cryptococcus, Epidermophyton, Fusarium, Kluyveromyces, Lachancea, Mucor, Neurospora, Ophiocoma, Penicillium, Pichia, Pneumocystis, Blastomyces, Saccharomyces, Schizosaccharomyces, Tolylcoceras, Trichoderma, Rhodotorula, or Yarrowia. In some embodiments, the cell is a Candida species cell, such as Candida albicans, Candida auris, Candida dubliniensis, Candida glabrata, Candida guillimonens, or Candida tropicalis cell. In some embodiments, the cell is a chytrid fungal cell, i.e., a cell of the phylum Chytridiomycetes. In some embodiments, the cell is a Batrachochytrium sp. cell, such as a Batrachochytrium cell. In certain embodiments, the cell is a microsporidian cell, such as a graminearum species or a microsporidian species cell. In certain embodiments, the fungal cell is a parasite. In certain embodiments, the cell is a member of a Trichophyton species or a Microsporum species cell, i.e., a member of the genus of fungi, including parasitic variants that cause tinea. In certain embodiments, the cell is a filamentous fungal cell, i.e., a cell from a filamentous fungus. In certain embodiments, the cell is a Cryptococcus species cell, such as a Cryptococcus neoformans cell. In certain embodiments, the cell is a Botrytis species cell, such as Botrytis cinerea, Botrytis welsh onion, Botrytis floridula, Botrytis ellipsoidea, Botrytis fabae, Botrytis scales, or Botrytis tracheiphila cell. iv. Other eukaryotic cells

[0210] In some embodiments, the eukaryotic cell comprising an HDR promoter is a microbial eukaryotic cell. In addition, the methods of the present disclosure can be used to increase HDR-mediated genome modification in a microbial eukaryotic cell, produce a microbial eukaryotic cell with a genome modification, and / or genetically engineer a microbial eukaryotic cell. In some embodiments, the methods disclosed herein include editing a microbial eukaryotic cell. In some embodiments, the methods disclosed herein include performing genome modification in a microbial eukaryotic cell. In some embodiments, the methods disclosed herein include modifying a target locus in a microbial eukaryotic cell. In some embodiments, the methods disclosed herein include increasing HDR-mediated genome modification in a microbial eukaryotic cell. In some embodiments, the cell is a microbial eukaryotic cell. In some embodiments, the cell is a cell of a unicellular eukaryotic organism. In some embodiments, the cell is a protozoan cell. In some embodiments, the cell is a protozoan cell. In some embodiments, the cell is an infectious microbial eukaryotic cell. In some embodiments, the cell is a parasitic microbial eukaryotic cell. In some embodiments, the cell is a Giardia species cell, such as Giardia lamblia, Giardia muris, Giardia heronii, Giardia psittaci, Giardia agile, or Giardia microti. In some embodiments, the cell is a Plasmodium species cell, such as Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, or Plasmodium knowlesi cell. In some embodiments, the cell is a kinetoplastid cell. In some embodiments, the cell is a Trypanosoma species cell, such as Trypanosoma krusei or Trypanosoma brucei cell.

[0211] In some embodiments, the cell is an algal cell. In some embodiments, the algal cell is of the following species: Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, hlamydomonas), Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, and Eremosphaera , Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, and Monopterus. Monoraphidium), Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella,In some embodiments, the cell is a diatom. Diatoms include members of the genera Achnanthes, Amphora, Chaetoceros, Coscinodiscus, Cylindrotheca, Cyclotella, Cymbella, Fragilaria, Fragilaropsis, Hantzschia, Navicula, Nitzschia, Pseudo-Nitzschia, Phaeodactylum, Psammodictyon, Skeletonema, Thalassionema, and Thalassiosira. In some embodiments, the cell is a eustigmatophyte, such as a species of the genus Nannochloropsis or a species of the genus Pseudostaurastrum, Vischeria, and Eustigmatos. In some embodiments, the cell is an algal cell of the genus Nannochloropsis, such as, but not limited to, N. gaditana, N. granulata, N. limnetica, N. oceanica,Eye spot microgreen algae (N.oculata), and salt marsh microgreen algae (N.salina).

[0212] In some embodiments, the cell is a long and short flagellate. For example, long and short flagellates include not only the true eye spot algae and diatoms listed above, but also chytrid species, including labrinthulids and thraustochytrids. In some embodiments, the cell belongs to long and short flagellates, including but not limited to Bacillariophytes, true eye spot algae, Labrinthulids, and thraustochytrids. In some embodiments, the cell belongs to the following species: Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Japonochytrium, Diplophrys, or Ulkenia. For example, the strain can be a species of Thraustochytrium, Schizochytrium, Oblongichytrium, or Aurantiochytrium. In some embodiments, the cell is an opisthokont. In some embodiments, the cell is a choanoflagellate. In some embodiments, the cell is a Mesomycetozoa (e.g., Sphaeroforma). In some embodiments, the cell is a unikont. In some embodiments, the cell is an amoebozoa. In some embodiments, the cell belongs to the genus Acanthamoeba, Amoeba, Chaos, Dictyostelium, Entamoeba, or Pelomyxa. v. Cell composition

[0213] Provided herein are compositions of cells. On the one hand, the methods provided herein can be used to produce compositions of eukaryotic cells. In some embodiments, the compositions of eukaryotic cells may be composed of any cell described herein, such as plants, animals, fungi or other eukaryotic cells. In some embodiments, the methods disclosed herein include editing cell populations. In some embodiments, the methods disclosed herein include producing edited cell populations, wherein the ratio of edited cells in the group is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, 30 times higher than the ratio of edited cells in the cell population edited in the absence of an HDR promoter, including any value or range between these values. In some embodiments, the methods disclosed herein include producing edited cell populations, wherein the ratio of edited cells in the group is 10 times higher than the ratio of edited cells in the cell population edited in the absence of an HDR promoter.

[0214] In some embodiments, provided herein are compositions of cells used in the methods provided herein, clonal subpopulations. In some embodiments, the clonal subpopulation is a subpopulation of a cell line. In some embodiments, the clonal subpopulation is a subpopulation of cells derived from an individual. In some embodiments, the clonal cell subpopulation is a cell population derived from a single cell. In some embodiments, the clonal cell subpopulation has the same genetic and epigenetic profiles.

[0215] In certain embodiments, the method disclosed herein includes performing genome modification in a cell group. In certain embodiments, the method disclosed herein includes producing a composition of cells with genome modification. In certain embodiments, the method disclosed herein includes producing a composition of cells with genome modification, wherein the ratio of cells with genome modification in the group is higher than the ratio of cells with genome modification in the modified cell group in the absence of an HDR promoter by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, 30 times, including any value or range between these values. In certain embodiments, the method disclosed herein includes modifying the target locus in a cell group. In certain embodiments, the method disclosed herein includes producing a cell group with a modified target locus. In some embodiments, the methods disclosed herein comprise generating a population of cells having a modified target locus, wherein the proportion of cells in the population having the modified target locus is greater than the proportion of cells having the modified target locus in the population of cells modified in the absence of an HDR promoter by about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, 30 times, including any value or range in between these values. E. Kit

[0216] The method of the present invention can be provided in the form of a kit. In some embodiments, the kit comprises a nucleic acid encoding an HDR promoter. In some embodiments, the kit comprises nucleic acids encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB) and instructions for use. In some embodiments, the kit provides a vector comprising nucleic acid. In some embodiments, the kit is used to modify the target editing site of a cell using a donor template DNA molecule. In some embodiments, the kit comprises any vector described herein. In some embodiments, the kit comprises a vector for increasing the HDR-mediated genomic modification of a target editing site of a eukaryotic cell genome (e.g., a plant or mammalian cell genome). In some embodiments, the kit comprises a vector for increasing the HDR-mediated genomic modification of a target editing site in a plant cell. In some embodiments, the kit comprises a vector for increasing the HDR-mediated genomic modification of a target editing site in a mammalian cell.

[0217] In some embodiments, the kit includes instructions. In some embodiments, the instructions include instructions for transforming cells with the nucleic acid. In some embodiments, the instructions include instructions for detecting the presence of the nucleic acid in the cell. In some embodiments, the instructions include instructions for evaluating the effect of the nucleic acid in the cell.

[0218] In some embodiments, the kit includes reagents for detecting genetically engineered cells. In some embodiments, the kit includes instructions for detecting genetically engineered cells using reagents. In some embodiments, the reagents for detecting genetically engineered cells are assays for assessing the cell genome, such as PCR assays, RT-qPCR assays, protein blotting, or sequencing assays. In some embodiments, the reagents for detecting genetically engineered cells are a set of oligonucleotide primers, some of which specifically amplify genetically modified or wild-type target loci. In some embodiments, detection of genetically engineered cells is performed using reporters, such as fluorescent reporters, transcriptional reporters, colorimetric reporters, or chemiluminescent reporters. Therefore, in some embodiments, the reagents for detecting genetically engineered cells are means for detecting reporter molecules.

[0219] In some embodiments, provided herein is a kit for increasing homology-directed repair (HDR)-mediated genome modification of a target editing site of a eukaryotic cell genome (e.g., a plant or mammalian cell genome). In some embodiments, the kit comprises nucleic acids encoding genome editing molecules and HDR promoters. In some embodiments, the genome editing molecule comprises: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cutting the DNA sequence at the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site. In some embodiments, the HDR promoter comprises a single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB). In some embodiments, the genome editing molecule and the HDR promoter thereby provide modification of the target editing site of the eukaryotic cell genome by a donor template polynucleotide at an increased frequency compared to a control by HDR. In some embodiments, the kit comprises a reagent for measuring the level of HDR-mediated genome modification at the target editing site.

[0220] In some embodiments, provided herein is a kit for preparing a eukaryotic cell with a genome modification. In some embodiments, the kit includes nucleic acids encoding genome editing molecules and homology-directed repair (HDR) promoters, wherein the genome editing molecules include: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule with homology to the target editing site, the at least one sequence-specific endonuclease cutting the DNA sequence at the target editing site; and wherein the HDR promoters include single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); thus, the genome editing molecules and the HDR promoters provide the modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide at an increased frequency compared to the control by HDR. In some embodiments, the kit provides a means for separating or propagating eukaryotic cells comprising genome modifications, thereby preparing eukaryotic cells with genome modifications. In some embodiments, the kit includes reagents for detecting the presence of genome modifications at target editing sites.

[0221] In some embodiments, provided herein are kits for methods of genetically engineering eukaryotic cells. In some embodiments, the kits comprise nucleic acids encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that at least partially converts a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). In some embodiments, the kits comprise reagents for detecting the genetic engineering status of the target editing site. Example

[0222] Various embodiments of eukaryotic cells (eg, plant cells and mammalian cells), systems, and methods provided herein are included in the following non-limiting list of examples. 1. A method for increasing homology-directed repair (HDR)-mediated genome modification of a target editing site in a eukaryotic cell genome, the method comprising: Providing genome editing molecules and HDR facilitators to a eukaryotic cell, wherein the genome editing molecules comprise: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site; and wherein the HDR facilitators comprise a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); The genome editing molecules and the HDR facilitators thereby provide for modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide via HDR at an increased frequency compared to a control. 2. The method of embodiment 1, 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. 3. The method of embodiment 2, wherein the RNA-guided nuclease comprises an RNA-guided DNA endonuclease, 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, or an engineered nuclease. 4. The method of embodiment 1, wherein the sequence-specific endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease. 5. The method of embodiment 1, wherein the genome editing molecules comprise one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleaves a single DNA strand at two different DNA sequences at the target editing site. 6. The method of embodiment 5, wherein the sequence-specific endonucleases comprise at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof. 7. The method of embodiment 5, wherein the sequence-specific endonucleases comprise Cas9 and / or Cas12a, and the guide RNA molecules have at least one base mismatch with the DNA sequence in the target editing site. 8. The method of embodiment 1, wherein the donor DNA molecule is provided on a circular DNA vector, a geminivirus replicon, or as a linear DNA fragment. 9. The method of any one of embodiments 1 to 8, wherein the donor DNA molecule is flanked by copies of an endonuclease recognition sequence. 10. The method of any one of embodiments 1 to 9, wherein the sequence-specific endonuclease comprises an RNA-guided nuclease and the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and immediately adjacent to a protospacer adjacent motif (PAM) sequence. 11. The method of any one of embodiments 1 to 10, wherein the sequence-specific endonuclease provides a 5' overhang at the target editing site after cleavage. 12. The method of any one of embodiments 1 to 11, wherein the SSAP provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules. 13. The method of any one of embodiments 1 to 12, wherein the SSAP comprises a RecT / Redβ family protein, an ERF family protein, or a RAD52 family protein. 14. The method of embodiment 13, wherein the RecT / Redβ family protein comprises a Rac bacterial prophage RecT protein, a phage λβ protein, a phage SPP1 35 protein, a related protein with equivalent SSAP activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, 2 or 3. 15. The method of embodiment 13, wherein the ERF family 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 to SEQ ID NO:4. 16. The method of embodiment 13, wherein the RAD52 family protein comprises 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 to SEQ ID NO: 5, 6 or 7. 17. The method of any one of embodiments 1 to 16, wherein linear dsDNA molecules are the preferred substrate for the exonuclease. 18. The method of embodiment 17, wherein linear dsDNA molecules comprising a phosphorylated 5' end are preferred substrates for the exonuclease. 19. The method of any one of embodiments 1 to 16, wherein the exonuclease has 5' to 3' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 20. The method of any one of embodiments 1 to 16, wherein the exonuclease has 3' to 5' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 21. The method of any one of embodiments 1 to 16, wherein the exonuclease comprises a bacteriophage lambda exo protein, a Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpes virus SOX protein, a UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, an exonuclease III, a Trex2 exonuclease, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144 or 145. 22. The method of any one of embodiments 1, 5, or 6, wherein the exonuclease comprises T7 phage exonuclease, E. coli exonuclease III, a related protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 143 or 144. 23. The method of any one of embodiments 1 to 22, wherein the single-stranded DNA binding protein (SSB) and the SSAP are obtained from the same host organism. 24. The method of any one of embodiments 1 to 23, wherein the single-stranded DNA binding protein (SSB) is a bacterial SSB or optionally an Enterobacteriaceae species SSB. 25. The method of embodiment 24, wherein the SSB is an Escherichia spp., Shigella spp., Enterobacter spp., Klebsiella spp., Serratia spp., Pantoea spp., or Yersinia spp. SSB. 26. The method of any one of embodiments 1 to 23, wherein the SSB comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 31, 34-131 or 132. 27. The method of any one of embodiments 1 to 26, wherein the frequency of HDR is increased by at least 2-fold compared to a control method in which control eukaryotic cells have the genome editing molecules but are not exposed to at least one of the HDR promoters. 28. The method of any one of embodiments 1 to 26, wherein the frequency of non-homologous end joining (NHEJ) is maintained or reduced by at least 2-fold compared to a control method in which control eukaryotic cells have the genome editing molecules but are not exposed to at least one of the HDR promoters. 29. The method of any one of embodiments 1 to 28, wherein the SSAP, the exonuclease and / or the SSB protein further comprises an operably linked nuclear localization signal (NLS) and / or a cell penetrating peptide (CPP). 30. The method of any one of embodiments 1 to 29, wherein the SSAP, the exonuclease, and / or the SSB are provided to the cell as a polyprotein comprising a protease recognition site or a self-processing protein sequence inserted between the SSAP, the exonuclease, and / or the SSB. 31. The method of any one of embodiments 1 to 30, wherein the eukaryotic cell is a mammalian cell or a plant cell. 32. The method of embodiment 31, wherein the plant cell is haploid, diploid, or polyploid. 33. The method of embodiment 32, wherein the plant cell is in culture, in a plant, or in a plant tissue. 34. The method of any one of embodiments 31-33, wherein the cell is a plant cell and the SSAP, the exonuclease and / or the single-stranded DNA binding protein further comprises an operably linked 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. 35. The method of any one of embodiments 31 to 34, further comprising the step of isolating and / or growing a plant cell, propagule, or plant obtained from the plant cell comprising the genomic modification, wherein the genome of the plant cell, propagule, or plant comprises the genomic modification. 36. A system for increasing homology-directed repair (HDR)-mediated genome modification of target editing sites in a eukaryotic cell genome, the system comprising: (a) Eukaryotic cells; (b) an HDR promoter comprising a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); and (c) one or more genome editing molecules comprising at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; wherein the eukaryotic cell is associated with, contacted with and / or contains: effective amounts of the HDR facilitators and the one or more genome editing molecules. 37. The system of embodiment 36, wherein the genome editing molecules and / or sequence-specific endonucleases comprise RNA-guided nucleases or polynucleotides encoding RNA-guided nucleases and guide RNAs or polynucleotides encoding guide RNAs. 38. The system of embodiment 37, wherein the RNA-guided nuclease comprises an RNA-guided DNA endonuclease, 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, or an engineered nuclease. 39. The system of embodiment 36, wherein the sequence-specific endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease. 40. The system of embodiment 36, wherein the genome editing molecules comprise one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleaves a single DNA strand at two different DNA sequences at the target editing site. 41. The system of embodiment 40, wherein the sequence-specific endonucleases comprise at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof. 42. The system of embodiment 40, wherein the sequence-specific endonucleases comprise Cas9 and / or Cas12a, and the guide RNA molecules have at least one base mismatch with the DNA sequence in the target editing site. 43. The system of embodiment 36, wherein the donor DNA molecule is provided on a plasmid or a geminivirus genome. 44. The system of any one of embodiments 36 to 43, wherein the donor DNA molecule is flanked by endonuclease recognition sequences. 45. The system of any one of embodiments 36 to 44, wherein the sequence-specific endonuclease comprises an RNA-guided nuclease and the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and immediately adjacent to the PAM sequence. 46. ​​The system of any one of embodiments 36 to 45, wherein the sequence-specific endonuclease provides a 5' overhang at the target editing site upon cleavage. 47. The system of any one of embodiments 36 to 46, whereby the genome editing molecules and the HDR facilitators provide modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide via HDR at a frequency that is at least 2-fold increased compared to a control. 48. The system of any one of embodiments 36 to 47, wherein the SSAP provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules. 49. The system of embodiment 36 or 48, wherein the SSAP comprises a RecT / Redβ family protein, an ERF family protein, or a RAD52 family protein. 50. The system of embodiment 49, wherein the RecT / Redβ family protein comprises a Rac bacterial prophage RecT protein, a bacteriophage λβ protein, a bacteriophage SPP1 35 protein, or a related protein having equivalent SSAP activity. 51. The system of embodiment 49, wherein the RecT / Redβ family protein comprises bacteriophage λβ protein, bacteriophage SPP1 35 protein, Rac bacterial prophage RecT protein, or a related protein with equivalent SSAP activity. 52. The system of embodiment 49, wherein the RecT / Redβ family protein comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, 2 or 3. 53. The system of embodiment 49, wherein the ERF family 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 to SEQ ID NO:4. 54. The system of embodiment 49, wherein the RAD52 family protein comprises 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 to SEQ ID NO: 5, 6 or 7. 55. The system of any one of embodiments 36 to 54, wherein linear dsDNA molecules are the preferred substrate for the exonuclease. 56. The system of any one of embodiments 36 to 54, wherein linear dsDNA molecules comprising a phosphorylated 5' end are preferred substrates for the exonuclease. 57. The system of any one of embodiments 36 to 54, wherein the exonuclease has 5' to 3' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 58. The system of any one of embodiments 36 to 54, wherein the exonuclease has 3' to 5' exonuclease activity and can recognize blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5' overhangs, and / or dsDNA substrates with 3' overhangs. 59. The system of any one of embodiments 36 to 58, wherein the exonuclease comprises a bacteriophage lambda exo protein, a Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpes virus SOX protein, a UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, an Escherichia coli exonuclease III, a mammalian Trex2 exonuclease, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144 or 145. 60. The system of any one of embodiments 36, 40, or 41, wherein the exonuclease comprises T7 phage exonuclease, E. coli exonuclease III, a related protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 143 or 144. 61. The system of any one of embodiments 36 to 60, wherein the single-stranded DNA binding protein (SSB) and the SSAP are obtained from the same host organism. 62. The system of any one of embodiments 36 to 61, wherein the single-stranded DNA binding protein (SSB) is a bacterial SSB or optionally an Enterobacteriaceae species SSB. 63. The system of embodiment 62, wherein the SSB is an Escherichia spp., Shigella spp., Enterobacter spp., Klebsiella spp., Serratia spp., Pantoea spp., or Yersinia spp. SSB. 64. The system of any one of embodiments 36 to 63, wherein the SSB comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 31, 34-131 or 132. 65. The system of any one of embodiments 36 to 64, wherein the frequency of HDR is increased at least 2-fold compared to a control system in which control eukaryotic cells have the genome editing molecules but are not exposed to at least one of the HDR promoters. 66. The system of any one of embodiments 36 to 64, wherein the frequency of non-homologous end joining (NHEJ) is maintained or reduced by at least 2-fold compared to a control system in which control eukaryotic cells have the genome editing molecules but are not exposed to at least one of the HDR promoters. 67. The system of any one of embodiments 36 to 66, wherein the SSAP, the exonuclease and / or the single-stranded DNA binding protein further comprises an operably linked nuclear localization signal (NLS) and / or a cell penetrating peptide (CPP). 68. The system of any one of embodiments 36 to 64, wherein the SSAP, the exonuclease and / or the SSB are provided to the cell as a polyprotein comprising a protease recognition site or a self-processing protein sequence inserted between the SSAP, the exonuclease and / or the SSB. 69. The system of any one of embodiments 36 to 68, wherein the eukaryotic cell is a mammalian cell or a plant cell. 70. The system of embodiment 69, wherein the plant cell is haploid, diploid, or polyploid. 71. The system of embodiment 69 or 70, wherein the plant cell is in culture, in a plant, or in a plant tissue. 72. The system of embodiment 69, 70 or 71, wherein the cell is a plant cell and the SSAP, the exonuclease and / or the single-stranded DNA binding protein further comprises an operably linked 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. 73. The system of any one of embodiments 69 to 72, wherein the system provides for isolating and / or growing a plant cell, propagule, or plant obtained from a plant cell comprising the genomic modification, and wherein the genome of the plant cell, propagule, or plant comprises the genomic modification. 74. A method for preparing a eukaryotic cell having a genome modification, the method comprising: (a) providing genome-editing molecules and homology-directed repair (HDR) facilitators to a eukaryotic cell, wherein the genome-editing molecules comprise: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease and a donor template DNA molecule having homology to the target editing site, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; and wherein the HDR facilitators comprise a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); thereby the genome-editing molecules and the HDR facilitators provide modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide via HDR at an increased frequency compared to a control; and (b) isolating or propagating eukaryotic cells comprising the genome modification, thereby preparing the eukaryotic cells having the genome modification. 75. The method of embodiment 74, wherein the genome editing molecules and / or sequence-specific endonucleases comprise RNA-guided nucleases or polynucleotides encoding RNA-guided nucleases and guide RNAs or polynucleotides encoding guide RNAs. 76. The method of embodiment 75, wherein the RNA-guided nuclease comprises an RNA-guided DNA endonuclease, 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, or an engineered nuclease. 77. The method of embodiment 74, wherein the sequence-specific endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease. 78. The method of embodiment 74, wherein the genome editing molecules comprise one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleave a single DNA strand at two different DNA sequences at the target editing site. 79. The method of embodiment 78, wherein the sequence-specific endonucleases comprise at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof. 80. The method of embodiment 78, wherein the sequence-specific endonucleases comprise Cas9 and / or Cas12a, and the guide RNA molecules have at least one base mismatch with the DNA sequence in the target editing site. 81. The method of embodiment 74, wherein the donor DNA molecule is provided in a plasmid or a geminivirus genome. 82. The method of any one of embodiments 74 to 81, wherein the donor DNA molecule is flanked by endonuclease recognition sequences. 83. The method of any one of embodiments 74 to 82, wherein the sequence-specific endonuclease comprises an RNA-guided nuclease and the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and immediately adjacent to the PAM sequence. 84. The method of any one of embodiments 74 to 83, wherein the sequence-specific endonuclease provides a 5' overhang at the target editing site upon cleavage. 85. The method of any one of embodiments 74 to 84, wherein the SSAP provides DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules. 86. The method of any one of embodiments 74 to 85, wherein the SSAP comprises a RecT / Redβ family protein, an ERF family protein, or a RAD52 family protein. 87. The method of embodiment 86, wherein the RecT / Redβ family protein comprises a Rac bacterial prophage RecT protein, a phage λβ protein, a phage SPP1 35 protein, or a related protein having equivalent SSAP activity. 88. The method of embodiment 86, wherein the RecT / Redβ family protein comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, 2 or 3. 89. The method of embodiment 86, wherein the ERF family 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 to SEQ ID NO:4. 90. The method of embodiment 86, wherein the RAD52 family protein comprises 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 to SEQ ID NO: 5, 6 or 7. 91. The method of any one of embodiments 74 to 90, wherein linear dsDNA molecules are the preferred substrate for the exonuclease. 92. The method of any one of embodiments 74 to 91, wherein linear dsDNA molecules comprising a phosphorylated 5' end are preferred substrates for the exonuclease. 93. The method of any one of embodiments 74 to 92, wherein the exonuclease has 5' to 3' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 94. The method of any one of embodiments 74 to 92, wherein the exonuclease has 3' to 5' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 95. The method of any one of embodiments 74 to 90, wherein the exonuclease comprises a bacteriophage lambda exo protein, a Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpes virus SOX protein, a UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, an Escherichia coli exonuclease III, a mammalian Trex2 exonuclease, a related protein with equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144 or 145. 96. The method of embodiment 74, 78 or 79, wherein the exonuclease comprises T7 phage exonuclease, Escherichia coli exonuclease III, a related protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 143 or 144. 97. The method of any one of embodiments 74 to 96, wherein the single-stranded DNA binding protein (SSB) and the SSAP are obtained from the same host organism. 98. The method of any one of embodiments 74 to 97, wherein the single-stranded DNA binding protein (SSB) is a bacterial SSB or optionally an Enterobacteriaceae species SSB. 99. The method of embodiment 98, wherein the SSB is an Escherichia species, Shigella species, Enterobacter species, Klebsiella species, Serratia species, Pantoea species, or Yersinia species SSB. 100. The method of any one of embodiments 74 to 99, wherein the SSB comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 31, 34-131 or 132. 101. The method of any one of embodiments 74 to 100, wherein the frequency of HDR is increased by at least 2-fold compared to a control method in which control eukaryotic cells have the genome-editing molecules but are not exposed to at least one of the HDR promoters. 102. The method of any one of embodiments 74 to 100, wherein the frequency of non-homologous end joining (NHEJ) is maintained or reduced by at least 2-fold compared to a control method in which control eukaryotic cells have the genome editing molecules but are not exposed to at least one of the HDR promoters. 103. The method of any one of embodiments 74 to 102, wherein the SSAP, the exonuclease and / or the single-stranded DNA binding protein further comprises an operably linked nuclear localization signal (NLS) and / or a cell penetrating peptide (CPP). 104. The system of any one of embodiments 74 to 103, wherein the SSAP, the exonuclease and / or the SSB are provided to the cell as a polyprotein comprising a protease recognition site or a self-processing protein sequence inserted between the SSAP, the exonuclease and / or the SSB. 105. The method of any one of embodiments 74 to 104, wherein the eukaryotic cell is a mammalian cell or a plant cell. 106. The method of embodiment 105, wherein the plant cell is haploid, diploid, or polyploid. 107. The method of embodiment 105 or 106, wherein the plant cell is in culture, in a plant, or in a plant tissue. 108. The method of embodiment 105, 106 or 107, wherein the SSAP, the exonuclease and / or the SSB further comprises an operably linked 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. 109. The method of any one of embodiments 105 to 108, further comprising the step of isolating and / or growing a plant cell, propagule, or plant obtained from the plant cell comprising the genomic modification, wherein the genome of the plant cell, propagule, or plant comprises the genomic modification. 110. The method of any one of embodiments 1 to 30, the system of any one of embodiments 36 to 68, or the method of any one of embodiments 74 to 104, wherein the HDR facilitators, genome editing molecules, and eukaryotic cells or eukaryotic cells comprising the genome modification are provided in an array comprising a plurality of containers, compartments, or positions, and wherein each container, compartment, or position comprises the HDR facilitators, genome editing molecules, and eukaryotic cells or eukaryotic cells comprising the genome modification. 111. A method for genetically engineering a eukaryotic cell, the method comprising providing to the eukaryotic cell: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in the eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB), The target editing site of the cell is modified by the donor template DNA molecule. 112. The method of embodiment 111, 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. 113. The method of embodiment 112, wherein the RNA-guided nuclease comprises an RNA-guided DNA endonuclease, 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, or an engineered nuclease. 114. The method of embodiment 111, wherein the sequence-specific endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease. 115. The method of embodiment 111, further comprising a guide RNA, wherein the sequence-specific endonucleases and the guide RNA cleave a single DNA strand at two different DNA sequences at the target editing site. 116. The method of embodiment 115, wherein the sequence-specific endonucleases comprise at least one Cas9 nickase, Cas12a nickase, zinc finger nickase, TALE nickase, or a combination thereof, wherein the sequence-specific endonuclease is specific for an endonuclease recognition sequence in the target editing site. 117. The method of embodiment 115, wherein the sequence-specific endonucleases comprise Cas9 and / or Cas12a, and the guide RNA molecules have at least one base mismatch with the DNA sequence in the target editing site. 118. The method of embodiment 111, wherein the donor DNA molecule is provided in a plasmid or a geminivirus genome. 119. The method of embodiment 111, wherein the donor DNA molecule is flanked by endonuclease recognition sequences. 120. The method of embodiment 111, wherein the SSAP comprises a RecT / Redβ family protein, an ERF family protein, or a RAD52 family protein. 121. The method of embodiment 120, wherein the RecT / Redβ family protein comprises a Rac bacterial prophage RecT protein, a phage λβ protein, a phage SPP1 35 protein, or a related protein having equivalent SSAP activity. 122. The method of embodiment 111, wherein linear dsDNA molecules are the preferred substrate for the exonuclease. 123. The method of embodiment 111, wherein linear dsDNA molecules comprising a phosphorylated 5' end are preferred substrates for the exonuclease. 124. The method of embodiment 111, wherein the exonuclease has 5' to 3' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 125. The method of embodiment 111, wherein the exonuclease has 3' to 5' exonuclease activity and can recognize a blunt-ended dsDNA substrate, a dsDNA substrate with an internal break in one strand, a dsDNA substrate with a 5' overhang, and / or a dsDNA substrate with a 3' overhang. 126. The method of embodiment 111, wherein the exonuclease comprises a bacteriophage lambda exo protein, a Rac prophage RecE exonuclease, an Artemis protein, an Apollo protein, a DNA2 exonuclease, an Exo1 exonuclease, a herpes virus SOX protein, a UL12 exonuclease, an enterobacterial exonuclease VIII, a T7 phage exonuclease, an Escherichia coli exonuclease III, a mammalian Trex2 exonuclease, a related protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8, 9, 136, 137, 138, 139, 140, 141, 142, 143, 144 or 145. 127. The method of embodiment 111, wherein the exonuclease comprises T7 phage exonuclease, Escherichia coli exonuclease III, a related protein having equivalent exonuclease activity, or a protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 143 or 144. 128. The method of embodiment 111, wherein the single-stranded DNA binding protein (SSB) and the SSAP are obtained from the same host organism. 129. The method of any one of embodiments 111 to 128, wherein the eukaryotic cell is a mammalian cell or a plant cell. 130. The method of embodiment 129, wherein the plant cell is haploid, diploid, or polyploid. 131. The method of embodiment 130, wherein the plant cell is in culture, in a plant, or in a plant tissue. 132. The method of embodiment 131, further comprising the step of isolating and / or growing a plant cell, propagule, or plant obtained from the plant cell comprising the genomic modification, wherein the genome of the plant cell, propagule, or plant comprises the genomic modification. 133. The method of any one of embodiments 111-132, wherein one or more of the i) at least one sequence-specific endonuclease, ii) the donor template DNA molecule having homology to the target editing site in the eukaryotic cell, iii) the single-stranded DNA annealing protein (SSAP), iv) the exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) the single-stranded DNA binding protein (SSB) are provided in one or more vectors. 135. The method of embodiment 133, wherein the vector is an Agrobacterium vector. 136. The method of any one of embodiments 111-132, wherein one or more of the i) at least one sequence-specific endonuclease, ii) the donor template DNA molecule having homology to the target editing site in the eukaryotic cell, iii) the single-stranded DNA annealing protein (SSAP), iv) the exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) the single-stranded DNA binding protein (SSB) is provided in a chromosome. 137. The method of any one of embodiments 111-132, wherein one or more of the i) at least one sequence-specific endonuclease, ii) the donor template DNA molecule having homology to the target editing site in the eukaryotic cell, iii) the single-stranded DNA annealing protein (SSAP), iv) the exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) the single-stranded DNA binding protein (SSB) is provided by introduction of a polypeptide, DNA, mRNA, and / or sexual hybridization. 138. The method of any one of embodiments 111-132, wherein one or more of the i) at least one sequence-specific endonuclease, ii) the donor template DNA molecule having homology to the target editing site in the eukaryotic cell, iii) the single-stranded DNA annealing protein (SSAP), iv) the exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) the single-stranded DNA binding protein (SSB) is provided by a progenitor cell comprising one or more of i)-v). wherein the progenitor cell does not comprise at least one of i)-v), wherein the at least one of i)-v) not comprised by the progenitor cell is subsequently provided by delivery of a polypeptide, DNA, or mRNA to the progenitor cell and / or sexual crossing of the progenitor cell. 139. The method of any one of embodiments 111-138, further comprising detecting the modification. 140. The method of embodiment 139, wherein detecting the modification comprises amplicon sequencing. 141. The method of any one of embodiments 111-140, wherein the target editing site is in a protein coding sequence or a promoter. 142. The method of any one of embodiments 111-141, wherein the modification of the target editing site is an insertion, deletion, or substitution. 143. The method of any one of embodiments 111-142, wherein the target editing site is a gene encoding an agronomically important trait or a gene involved in a mammalian disease. 144. A method for producing a eukaryotic cell having a genetically modified target editing site, the method comprising: (a) providing at least one sequence-specific endonuclease or at least one polynucleotide encoding said at least one sequence-specific endonuclease, which at least one sequence-specific endonuclease cleaves the DNA sequence of at least one endonuclease recognition sequence in said target editing site, and (b) providing at least one donor molecule comprising at least one double-stranded DNA sequence, wherein (i) the DNA sequence is at least 90% homologous to a sequence flanking the target editing site over a length of at least 50 nucleotides and (ii) the donor sequence comprises at least one modification compared to the target editing site; and (c) providing at least one homology-directed repair (HDR) facilitator comprising (i) at least one single-stranded DNA annealing protein (SSAP), and (ii) at least one exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and (iii) at least one single-stranded DNA binding protein (SSB); and whereby the at least one sequence-specific endonuclease, the at least one donor molecule, and the at least one HDR facilitator introduce said modification into said target editing site of said eukaryotic cell; and (d) isolating a eukaryotic cell comprising a modification in the target editing site. 145. The method of embodiment 144, wherein the modification is selected from the group consisting of: insertion of one or more nucleotides, deletion of one or more nucleotides, or substitution of one or more nucleotides. 146. The method of embodiment 144, wherein a portion of the target editing site is deleted using two sequence-specific cuts in the target editing site and replaced by the sequence provided by the donor molecule. 147. The method of any one of embodiments 144-146, wherein the donor sequence is flanked by endonuclease recognition sequences in the vector. 148. The method of any one of embodiments 144-147, further comprising propagating the eukaryotic cell comprising the modification. 149. A method for producing a genetically modified organism, the method comprising the steps of (i) producing a genetically modified eukaryotic cell as described in any one of Examples 144-148, and (ii) regenerating the cell into an organism. 150. The organism of embodiment 149, wherein the organism is selected from the group consisting of plants and non-human animals. 151. A composition comprising nucleic acids encoding one or more of: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). 152. The composition of embodiment 151, wherein the nucleic acids are located in one or more vectors. 153. A vector comprising a nucleic acid encoding one or more of: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). 154. The vector of embodiment 153, wherein the vector comprises the donor template DNA, the sequence-specific endonuclease, and the polynucleotide encoding the guide RNA. 155. The vector of embodiment 153, wherein the vector comprises the single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and the single-stranded DNA binding protein (SSB). 156. The vector of embodiment 153, wherein the vector comprises nucleic acids encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). 157. A kit comprising nucleic acids encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that at least partially converts a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB) and instructions for use in genetically engineering a eukaryotic cell. 158. The kit of embodiment 157, wherein the kit comprises a first vector and a second vector, wherein i) the first vector comprises a nucleic acid comprising the donor template DNA and the sequence-specific endonuclease, wherein the sequence-specific endonuclease comprises a polynucleotide encoding an RNA-guided nuclease and a polynucleotide encoding a guide RNA; and ii) the second vector comprises the single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and the single-stranded DNA binding protein (SSB). 159. The kit of any one of embodiments 157-158, further comprising reagents for detecting genetically engineered cells. 160. A cell comprising i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). 161. A cell comprising a nucleic acid encoding: i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule having homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). 162. The cell of embodiment 160 or 161, wherein the cell is a plant or mammalian cell. 163. The cell of any one of embodiments 160-162, wherein the cell is a host cell. 164. A genetically engineered cell produced by the method of any one of embodiments 1-35 or 74-149. 165. A progenitor eukaryotic cell or organism for genetic engineering at a target editing site, the progenitor eukaryotic cell or organism comprising a subset of: i) at least one sequence-specific endonuclease, ii) a donor template molecule having homology to the target editing site in the eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease that is capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB), wherein the cell does not comprise at least one of i)-v), wherein providing the cell or organism with at least one of i)-v) not comprised in the progenitor cell or organism results in modification of the target editing site by the donor template molecule. 166. The ancestral eukaryotic cell or ancestral organism of embodiment 165, wherein the donor template is a double-stranded DNA molecule. 167. A progenitor cell as described in Example 165, wherein the cell is a germline cell. 168. The progenitor eukaryotic cell or progenitor organism of embodiment 165, wherein the progenitor eukaryotic cell is a progenitor plant cell, and at least one of i)-v) not comprised by the progenitor plant cell or plant is provided by transformation. 169. The progenitor organism of embodiment 165, wherein the organism is a plant, and wherein at least one of i)-v) not contained by the progenitor plant is provided by sexual hybridization with a second plant containing at least one of i)-v) not contained by the progenitor plant. 170. The progenitor eukaryotic cell of embodiment 165, wherein the progenitor eukaryotic cell is an animal cell, and wherein at least one of i)-v) not contained in the progenitor cell is provided by transfection. 171. The progenitor organism of embodiment 165, wherein the progenitor organism is a non-human animal and at least one of i)-v) not contained by the non-human animal is provided by sexual hybridization with a non-human animal containing at least one of i)-v) not contained by the non-human animal. 172. The vector of embodiment 153, wherein the sequence-specific nuclease is operably linked to an inducible promoter. 173. The method of embodiment 111, wherein the sequence-specific endonuclease is a nicking enzyme. Examples

[0223] The following examples are intended to be purely exemplary of the present invention and therefore should not be considered to limit the present invention in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation. Example 1. Exonucleases, SSAP and SSB Expression Vectors and Donor DNA Template Sequences

[0224] This example describes the construction of plant expression vectors for the expression of bacteriophage lambda exonuclease (SEQ ID NO: 8), bacteriophage lambda beta SSAP protein (SEQ ID NO: 1), and E. coli SSB (SEQ ID NO: 31).

[0225] Plant expression constructs were constructed for the expression of bacteriophage lambda exonuclease (SEQ ID NO: 8), bacteriophage lambda beta SSAP protein (SEQ ID NO: 1), and E. coli SSB (SEQ ID NO: 31). A DNA sequence encoding the tobacco c2 nuclear localization signal (NLS) of SEQ ID NO: 15 was operably linked to DNA sequences encoding the exonuclease, bacteriophage lambda beta SSAP protein, and E. coli SSB to provide DNA sequences encoding the c2 NLS-Exo (also known as Red-Exo), c2 NLS lambda beta SSAP (also known as Red-beta), and c2 NLS-SSB fusion proteins set forth in SEQ ID NO: 135, SEQ ID NO: 134, and SEQ ID NO: 133, respectively. The DNA sequences encoding c2 NLS-Exo, c2 NLSλβSSAP, and c2NLSf-SSB fusion proteins were operably linked to the 2x35S, SlUBI10, and PcUBI4 promoters and the 35S, AtHSP, and pea3A polyadenylation sites, respectively, to provide exonuclease, SSAP, and SSB plant cell gene expression cassettes (see Figure 2 ).

[0226] A DNA donor template plasmid targeting the promoter region of the tomato Ant1 gene was constructed to insert a 42 base pair heterologous sequence via HDR ( Figure 1). The circular DNA donor plasmid includes a replacement template having the desired insertion region (42 base pairs long) flanked by homology arms of approximately 600-800 bp in length. The homology arms match (i.e., are homologous to) the gDNA (genomic DNA) regions flanking the target gDNA insertion site. The replacement template region comprising the donor DNA is flanked at each end by DNA sequences identical to the target gDNA sequence recognized by the RNA-guided nuclease. A plant expression cassette is also constructed for expressing an RNA-guided sequence-specific endonuclease and a guide RNA complementary to a sequence adjacent to the insertion site. Figure 1 ). Example 2. Genome editing experiment in tomato protoplasts

[0227] This example describes gene editing in tomato protoplasts using blunt- and staggered-end-cutting CAS nucleases in the presence and absence of the exonucleases, SSB and SSAP.

[0228] Essentially according to the published procedure ( et al. 2017) isolation, culture, and PEG-mediated transfection of tomato protoplasts. The transfected materials included plasmids with the donor DNA template region described in Example 1, and expression of gRNA and Cas polynucleotides as indicated ( Figure 1 The Cas polynucleotide is fused to a nuclear localization signal. The gRNA targets the double-strand break into the intended genomic DNA target and releases the replacement template from the donor plasmid (see Figure 1 Some experiments were performed with the following Cas nuclease, which represents a Cas nuclease that leaves blunt ends after cleaving the endonuclease recognition sequence and is referred to herein as CasB nuclease. Other experiments were performed with the following Cas nuclease, which represents a Cas nuclease that leaves staggered single-stranded DNA overhangs after cleaving the endonuclease recognition sequence and is referred to herein as CasS nuclease.

[0229] After 48 h of incubation of protoplasts after transfection, gDNA was extracted from the transfected samples and the target locus was amplified using primers complementary to the genomic sequences flanking the introduced replacement sequence and the homology arms of the replacement template and analyzed by amplicon sequencing.

[0230] The amplicon was sequenced using double-end Illumina sequencing. Due to the size of the amplicon, only one read end (read 1) in the double-end read covered the target site containing the targeted sequence insertion. The target read (read 1) was quality trimmed and aligned with the reference amplicon. These reads have unique molecular identifier (UMI) tags to distinguish them from certain types of PCR repeats, and these reads are de-duplicated from the alignment. The read (read 2) mapped to the unedited genomic sequence was then checked for correct mapping to the genome. The alignment generated from read 1 was analyzed using CrispRVariants, which describes and records all sequence alleles that are different within a 100bp window centered on the cut site (Lindsay, H. et al. Nature Biotechnology 2016 34:701-702). CrispRVariants reports the read frequency of each allele in terms of the number of reads of the total alignment. Different sequence alleles are classified as 1) wild-type sequence, SNP or sequencing artifact,...

Claims

1. A method for increasing homology-directed repair (HDR)-mediated genome modification of a target editing site in a eukaryotic cell genome, the method comprising: Providing genome-editing molecules and an HDR facilitator to a eukaryotic cell, wherein the genome-editing molecules comprise: (i) at least one sequence-specific endonuclease or at least one polynucleotide encoding the sequence-specific endonuclease, the at least one sequence-specific endonuclease cleaving the DNA sequence at the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site; and These HDR promoters include single-stranded DNA annealing protein (SSAP), an exonuclease that can at least partially convert a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); The genome editing molecules and the HDR facilitators thereby provide for modification of the target editing site of the eukaryotic cell genome by the donor template polynucleotide via HDR at an increased frequency compared to a control.

2. The method of claim 1, 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.

3. The method of claim 2, wherein the RNA-guided nuclease comprises an RNA-guided DNA endonuclease, 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, or an engineered nuclease.

4. The method of claim 1, wherein the sequence-specific endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease.

5. The method of claim 1 , wherein the genome editing molecules comprise one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleave a single DNA strand at two different DNA sequences at the target editing site.

6. The method of claim 5, wherein the sequence-specific endonucleases comprise at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof.

7. The method of claim 5, wherein the sequence-specific endonucleases comprise Cas9 and / or Cas12a, and the guide RNA molecules have at least one base mismatch with the DNA sequence in the target editing site.

8. The method of claim 1, wherein the donor DNA molecule is provided on a circular DNA vector, a geminivirus replicon, or as a linear DNA fragment.

9. The method of claim 1, wherein the donor DNA molecule is flanked by copies of an endonuclease recognition sequence.

10. The method of claim 1, wherein the sequence-specific endonuclease comprises an RNA-guided nuclease and the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and immediately adjacent to a protospacer adjacent motif (PAM) sequence.

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