Conceptio › Archive › Google Patents

Google Patentsopen access

Methods for improved plant gene-editing — Inari Agriculture Technology, Inc. (US12043838B2)

Inari Agriculture Technology, Inc. · Google Patents

Google Patents · Patents · License: Open Access

Open Source ↗

patent, google patents, intellectual property, US12043838B2, Inari Agriculture Technology, Inc., Hannah PHAM, en, 2024

ABSTRACT

Abstract

Systems, methods, and compositions for providing improvements in plant gene-editing efficiency are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation patent application of U.S. Ser. No. 16/963,372, filed Jul. 20, 2020, which is a National Phase Application of PCT/US2019/014559, filed on Jan. 22, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/783,301, filed Dec. 21, 2018, and U.S. Provisional Patent Application No. 62/620,130, filed Jan. 22, 2018, all of which are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on Mar. 2, 2023, is named âP13449US03_SequenceListing.xmlâ and is 1,163,887 bytes in size.

BACKGROUND

Recent advances in genome editing technologies have provided opportunities for precise modification of the genome in many types of organisms, including plants and animals. For example, technologies based on genome editing proteins, such as zinc finger nucleases, TALENs, and CRISPR systems are advancing rapidly and it is now possible to target genetic changes to specific DNA sequences in the genome. Methods for growing and manipulating plant cells, embryos, callus tissue, plant protoplasts, and plants are especially useful for genome editing as well as genetic engineering technologies.

SUMMARY

Disclosed herein are systems and methods for editing target genes in plant cells. Also disclosed are compositions that provide for editing target genes in plant cells.

In one aspect, systems for modification of a plant genome and/or target plant gene are provided. In certain embodiments, the systems for modification of a plant gene comprise: (a) a plant cell grown under a hypoxic condition, or treated with a reactive oxygen species (ROS) scavenging agent, or both grown under the hypoxic condition and treated with the ROS scavenging agent; (b) at least one of an exogenous, heterologous, and/or overproduced plant cell synthesis phase (S-phase) promoting agent, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; and (c) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell grown under the hypoxic condition is associated with, contacts, and/or contains said S-phase promoting agent, homology-dependent repair promoting agent, and/or non-homologous end-joining (NHEJ) inhibitory agent, and said molecule(s) or wherein said plant cell treated with the ROS scavenging agent is associated with, contacts, and/or contains said S-phase promoting agent, homology-dependent repair promoting agent, and/or a non-homologous end-joining (NHEJ) inhibitory agent, said ROS scavenging agent, and said molecule(s). In certain embodiments, the systems for modification of a plant gene comprise: (a) a plant cell wherein a reactive oxygen species (ROS) concentration is lowered in comparison to a control plant cell; (b) at least one of an exogenous, heterologous, and/or overproduced plant cell synthesis phase (S-phase) promoting agent, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; and (c) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell is associated with, contacts, and/or contains said agent(s) and said molecule(s). Systems for modification of a plant gene comprising: (a) a plant cell; (b) a plant cell synthesis phase (S-phase) promoting agent; (c) a homology-dependent repair promoting agent and/or a non-homologous end-joining (NHEJ) inhibitory agent; and (d) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell is associated with, contacts, and/or contains one or more of said agents and said molecule(s) are also provided. Uses of the aforementioned systems to modify a plant genome and/or a target plant gene are also provided herein.

Methods for modifying a plant cell genome or a target plant gene in a plant cell genome are also provided. In certain embodiments, the methods for modifying a plant cell genome or a target plant gene in a plant cell genome comprise: (a) providing genome editing molecules to a plant cell exposed to (i) a hypoxic growth condition, a reactive oxygen species (ROS) concentration lowering agent, or combination thereof; and (ii) at least one of an exogenous, heterologous, and/or overproduced plant cell synthesis phase (S-phase) promoting agent, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; wherein the molecules comprise: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof, wherein the molecules modify the plant cell genome. Methods for modifying a plant cell genome comprising: (a) providing genome editing molecules to a plant cell previously, concurrently, or subsequently exposed to: (i) a plant cell synthesis phase (S-phase) promoting agent; and (ii) at least one of a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof, wherein the molecules comprise: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof, wherein the molecules modify the plant cell genome, are also provided.

Compositions, plant cell cultures, and/or reaction mixtures comprising plant cells, certain agents, and gene editing molecules are also provided herein. In certain embodiments, the compositions, plant cell cultures, or reaction mixtures comprise: (a) a plant cell grown under a hypoxic condition, treated with an exogenous reactive oxygen species (ROS) scavenging agent, or both grown under the hypoxic condition and treated with the ROS scavenging agent; or a plant cell or plant cell grown under a hypoxic condition and an exogenous ROS scavenging agent; (b) at least one of an exogenous, heterologous, and/or overproduced plant cell synthesis phase (S-phase) promoting agent, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; and (c) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell grown under the hypoxic condition is associated with, contacts, and/or contains said S-phase promoting agent, homology-dependent repair promoting agent, and/or non-homologous end-joining (NHEJ) inhibitory agent, and said molecule(s), wherein said plant cell treated with the ROS scavenging agent is associated with, contacts, and/or contains said S-phase promoting agent, said ROS scavenging agent, and said molecule(s), or wherein said plant cell or plant cell grown under a hypoxic condition is associated with, contacts, and/or contains said S-phase promoting agent, homology-dependent repair promoting agent, and/or non-homologous end-joining (NHEJ) inhibitory agent, said ROS scavenging agent, and said molecule(s). In certain embodiments, the compositions, plant cell cultures, or reaction mixtures comprise: (a) a plant cell wherein a reactive oxygen species (ROS) concentration is lowered in comparison to a control plant cell; (b) at least one of an exogenous plant cell synthesis phase (S-phase) promoting agent; a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; and (c) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell is associated with, contacts, and/or contains said agent(s) and said molecule(s). In certain embodiments, the compositions, plant cell cultures and/or reaction mixtures comprise: (a) a plant cell; (b) an exogenous plant cell synthesis phase (S-phase) promoting agent (c) at least one of a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; and (d) genome editing molecule(s) comprising: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (iv) one or more polynucleotide(s) encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof; wherein said plant cell is associated with, contacts, and/or contains said agent and said molecule(s). Uses of the aforementioned compositions, plant cell cultures, or reaction mixtures to modify a plant genome and/or a target plant gene are also provided herein.

Methods are provided for making a plant cell having a genomic modification comprising: (a) providing genome editing molecules to a plant cell previously, concurrently, or subsequently exposed to: (i) a hypoxic growth condition, a reactive oxygen species (ROS) concentration lowering agent, or combination thereof; and (ii) at least one of an exogenous, heterologous, and/or overproduced plant cell synthesis phase (S-phase) promoting agent, a homology-dependent repair promoting agent, a non-homologous end-joining (NHEJ) inhibitory agent, or any combination thereof; wherein the molecules comprise: (i) an RNA-guided nuclease and a guide RNA and optionally a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof, to modify the plant cell's genome; and, (b) isolating or propagating a plant cell comprising the genome modification, thereby making the plant cell having a genomic modification. Methods are also provided for making a plant cell having a genomic modification comprising: (a) providing genome editing molecules to a plant cell previously, concurrently, or subsequently exposed to: (i) a plant cell synthesis phase (S-phase) promoting agent; and (ii) a homology-dependent repair promoting agent and/or a non-homologous end-joining (NHEJ) inhibitory agent, wherein the molecules comprise: (i) an RNA-guided nuclease and a guide RNA; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding a RNA-guided nuclease and a guide RNA; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof, to modify the plant cell's genome; and, (b) isolating or propagating a plant cell comprising the genome modification, thereby making the plant cell having a genomic modification. In certain embodiments, the methods can further comprise obtaining callus, a propagule, or a plant from the isolated or propagated plant cell of step (b) comprising the genome modification, wherein the callus, propagule, or plant comprises a genome modified by the molecule(s). In certain embodiments, the propagule is a seed or the methods further comprise obtaining a seed from the plant, said seed comprising the genome modification.

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5â² to 3â² direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate embodiments described by the plural of that term.

The term âand/orâ where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/orâ as used in a phrase such as âA and/or Bâ herein is intended to include âA and B.â âA or B.â âAâ (alone), and âBâ (alone). Likewise, the term âand/orâ as used in a phrase such as âA, B, and/or Câ is intended to encompass 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).

As used herein, the term âcontrolâ refers to a reference standard where one or more treatments are omitted.

As used herein, the phrase âgene-editingâ includes genome modification by homology directed repair (HDR) mechanisms. Such gene-editing includes embodiments where a site specific (sequence specific) nuclease and a donor template are provided.

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

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

As used herein, the terms âinclude,â âincludes,â and âincludingâ are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

As used herein, there terms âorthologâ or âorthologousâ refer to genes and/or encoded proteins from different species that have the similar or identical functions (e.g., exhibit similar or identical phenotypes when suppressed and/or overexpressed). Orthologous genes and their encoded proteins will typically exhibit a certain degree of sequence conservation and a similar pattern of expression (e.g., tissue, temporal, and/or cell cycle stage specific expression). Sequence conservation in orthologous genes and their encoded proteins can extend over the entire polynucleotide or amino acid sequence or can be limited to certain functional domains (e.g., transcription activation, DNA, protein, substrate, and or membrane binding, dimerization, oligomerization) of the encoded protein or residues located therein and the corresponding polynucleotide coding sequence. In certain embodiments, sequence conservation in an orthologous gene to the gene can be at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% over the entire polynucleotide or amino acid sequence or to portions of a polynucleotide or amino acid sequence which respectively encode or comprise certain functional domains (e.g., pRb binding motif or domain including an LXCXE motif and/or a helix 4 motif).

As used herein, the term âoverproducedâ where used herein with regards to various agents refers to providing the agent in an amount that is increased in comparison to the amount found i

CROSS-REFERENCE TO RELATED APPLICATIONS

INCORPORATION OF SEQUENCE LISTING

BACKGROUND

SUMMARY

Disclosed herein are systems and methods for editing target genes in plant cells. Also disclosed are compositions that provide for editing target genes in plant cells.

DETAILED DESCRIPTION

As used herein, the term âcontrolâ refers to a reference standard where one or more treatments are omitted.

As used herein, the terms âinclude,â âincludes,â and âincludingâ are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

As used herein, the term âoverproducedâ where used herein with regards to various agents refers to providing the agent in an amount that is increased in comparison to the amount found in an untreated plant cell or plant.

As used herein, the phrase âoxygen speciesâ refers to both oxygen (O 2 ) and reactive oxygen species (ROS). The phrase âReactive Oxygen Speciesâ refers to radical and non-radical oxygen species formed by the partial reduction of oxygen. Examples of ROS include hydrogen peroxide, a superoxide radical, a peroxide ion, a hydroperoxyl radical, and/or a hydroxyl radical.

The term âpolynucleotideâ where used herein is a nucleic acid molecule containing 2 or more nucleotide residues. Polynucleotides are generally described as single- or double-stranded. Where a polynucleotide contains double-stranded regions formed by intra- 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 can employ or include: (i) one or more polynucleotides of 2 to 25 residues in length, one or more polynucleotides of more than 26 residues in length, or a mixture of both. Polynucleotides can comprise single- or double-stranded RNA, single- or double-stranded DNA, double-stranded DNA/RNA hybrids, chemically modified analogues thereof, or a mixture thereof. In certain embodiments, a polynucleotide can include a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or can includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes 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 a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope). Polynucleotides provided or used herein also include modified nucleic acids, particularly modified RNAs, which are disclosed in U.S. Pat. No. 9,464,124, which is incorporated herein by reference in its entirety.

As used herein, the term âRepAâ or âRepA proteinâ includes plant geminivirus proteins designated AL1, AC1, C1, or RepA that bind to and inhibit a plant retinoblastoma protein.

As used herein, the phrase âretinoblastoma proteinâ or term âpRbâ refers to the same gene or protein referred to by the phrase âRETINOBLASTOMA-RELATEDâ or term âRBRâ, as well as orthologs of the Arabidopsis RBR gene or protein. A representative dicot plant pRB or RBR sequence is provided herewith as SEQ ID NO:22. Representative monocot plant pRB or RBR sequences are provided herewith as SEQ ID NO: 29, 30, and 31.

As used herein the term âsynergisticâ refers to an effect of combining at least two factors that exceeds the sum of the effects obtained when the factors are not combined.

As used herein, the phrase âsynthesis phaseâ and term âS-phaseâ both refer to the stage of the plant cell cycle where the plant cell DNA is replicated. The S-phase occurs after the G 1 phase and before the G 2 phase of the cell cycle.

As used herein, the phrase âtarget plant geneâ refers to a gene located in the plant genome that is to be modified by gene editing molecules provided in a system, method, composition and/or plant cell provided herein. Embodiments of target plant genes include (protein-)coding sequence, non-coding sequence, and combinations of coding and non-coding sequences. Modifications of a target plant gene include nucleotide substitutions, insertions, and/or deletions in one or more elements of a plant gene that include a transcriptional enhancer or promoter, a 5â² or 3â² untranslated region, a mature or precursor RNA coding sequence, an intron, a splice donor and/or acceptor, a protein coding sequence, a polyadenylation site, and/or a transcriptional terminator. 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 of the modified target gene in the plant cell. In embodiments, where a desired trait is conferred by a loss-of-function mutation that is introduced into the target gene by gene editing, a plant cell, population of plant cells, plant, or seed is homozygous for a modified target gene with the loss-of-function mutation. In other embodiments, only a subset of the copies or alleles of a given target gene are modified to provide heterozygosity of the modified target gene in the plant cell. In certain embodiments where a desired trait is conferred by a dominant mutation that is introduced into the target gene by gene editing, a plant cell, population of plant cells, plant, or seed is heterozygous for a modified target gene with the dominant mutation. Traits imparted by such modifications to certain plant target genes include improved yield, resistance to insects, fungi, bacterial pathogens, and/or nematodes, herbicide tolerance, abiotic stress tolerance (e.g., 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, and the like, all in comparison to a control plant that lacks the modification. The plant having a genome modified by gene editing molecules provided in a system, method, composition and/or plant cell provided herein differs from a plant having a genome modified by traditional breeding (i.e., crossing of a male parent plant and a female parent plant), where unwanted and random exchange of genomic regions as well as random mitotically or meiotically generated genetic and epigenetic changes in the genome typically occurs during the cross and are then found in the progeny plants. Thus, in embodiments of the plant (or plant cell) with a modified genome, the modified genome is more than 99.9% identical to the original (unmodified) genome. In embodiments, the modified genome is devoid of random mitotically or meiotically generated genetic or epigenetic changes relative to the original (unmodified) genome. In embodiments, the modified genome includes a difference of epigenetic changes in less than 0.01% of the genome relative to the original (unmodified) genome. In embodiments, the modified genome includes: (a) a difference of DNA methylation in less than 0.01% of the genome, relative to the original (unmodified) genome; or (b) a difference of DNA methylation in less than 0.005% of the genome, relative to the original (unmodified) genome; or (c) a difference of 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 the plant cell, and the modified genome includes: (a) a difference of DNA methylation in less than 0.01% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome; or (b) a difference of DNA methylation in less than 0.005% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome; or (c) a difference of DNA methylation in less than 0.001% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome. In embodiments, the modified genome has not more unintended changes in comparison to the original (unmodified) genome than 1Ã10{circumflex over (â)}â8 mutations per base pair per replication.

To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

Systems, methods, and compositions that provide for increased frequencies of plant gene editing in comparison to controls are provided herein. Such systems, methods and compositions can comprise a combination at least two features that provide for such increased plant gene editing frequencies. In certain embodiments, a first feature comprises plant cells that have been exposed to hypoxic conditions and/or agents that reduce reactive oxygen species (ROS) or plant cells that have lowered ROS concentrations. In certain embodiments, a second feature comprises treatment of the plant cells that have been exposed to hypoxic conditions and/or agents that reduce ROS or the plant cells that have lowered ROS concentrations with a plant cell synthesis phase (S-phase) promoting agent. In certain embodiments, this combination of features has been shown to provide an increase of gene editing frequency through homology directed repair (HDR) pathways that exceed increases provided by either of the features alone or provided by the sum of the increases provided by each of the features. In certain embodiments, this combination of elements has been shown to provide a synergistic increase in frequencies of gene editing through homology directed repair (HDR) pathways that exceeds the sum of the increases in HDR provided by each of the features alone.

In certain embodiments of the systems, methods, and compositions provided herein, the plant cell is exposed to and/or maintained under hypoxic conditions. Normal (i.e., ânormoxicâ) oxygen conditions comprise about 20% oxygen by volume. Hypoxic conditions used in the systems, methods, and compositions provided herein can in certain embodiments comprise about 14%, 13%, 12%, 11%, or 10% to about 8%, 7%, 6%, or 5% oxygen by volume. In certain embodiments, hypoxic conditions can comprise treating the plant cells with a hypoxia mimetic (e.g., desferrioxamine or cobalt chloride). In certain embodiments, a hypoxic condition can comprises maintaining the cell in a liquid culture media having a dissolved oxygen concentration that is lower than the dissolved oxygen concentration obtained when the liquid culture media is under normoxic conditions. Such exposure of the plant cell to the hypoxic condition can in certain embodiments be limited to a period of time necessary to realize improvements in gene editing frequencies (e.g., prior to and/or during association, contact, and/or containment to/of an S-phase promoting agent and/or gene editing molecule; prior to and/or during exposure and/or after to an S-phase promoting agent and/or gene editing molecule). Such exposure and or maintenance of a plant cell under hypoxic conditions can be achieved in the context of a plant cell in isolated form (e.g., as a protoplast), a plant cell in a plant embryo, plant callus, especially embryogenic callus, in an isolated plant tissue or part (e.g., an ovule, anther, leaf, meristematic tissue, and the like), or in a whole plant. In certain embodiments, the plant cell in any of the aforementioned contexts can be in a liquid or solid culture medium that includes about 20, about 40, or about 60 to about 80, about 100, about 120, or about 150 millimolar Ca 2+ and/or Mg 2+ , and is exposed to and/or maintained under hypoxic conditions. In certain embodiments, the plant cells (e.g., plant protoplasts) are exposed to the hypoxic conditions about 5, 10, 15, 30, or 45 minutes to about 60, 75, 90, or 120 minutes after exposure to the gene-editing molecules and/or S-phase promoting agent. In certain embodiments, the combination of the aforementioned hypoxic conditions with an S-phase promoting agent provides a synergistic increase in frequencies of gene editing through homology directed repair (HDR) pathways that exceeds the sum of the increases in HDR provided by the hypoxic conditions and S-phase promoting agents alone. In certain embodiments, the combination of the aforementioned hypoxic conditions with an S-phase promoting agent and any of the aforementioned divalent cations provides a synergistic increase in frequencies of gene editing through homology directed repair (HDR) pathways that exceeds the sum of the increases in HDR provided each of the hypoxic conditions, S-phase promoting agents, and divalent cations alone.

Embodiments of the systems, methods, or compositions provided herein include cultures wherein the plant cell is exposed or treated with an enzymatic and/or a non-enzymatic ROS scavenging agent. In certain embodiments, such exposure or treatment with the enzymatic and/or a non-enzymatic ROS scavenging agent results in lowered concentrations of ROS (e.g., hydrogen peroxide, a superoxide radical, a peroxide ion, a hydroperoxyl radical, and/or a hydroxyl radical) in the exposed or treated plant cell in comparison to an unexposed or untreated plant cell. In certain embodiments, the non-enzymatic ROS scavenging agents include low-molecular-weight antioxidants, including lipid-soluble antioxidants and water-soluble antioxidants (e.g., low-molecular-weight thiol antioxidants, pro-thiols, ascorbic acid, tocopherols, carotenoids, flavonoids, butylated hydroxytoluene, and butylated hydroxyanisole). In certain embodiments, the non-enzymatic ROS scavenging agents are provided at a concentration of about 0.1 to about 10 millimolar. Specific embodiments include cultures wherein the culture medium includes about 0.1 to about 10 millimolar low-molecular-weight thiol antioxidants; sec, e.g., Pivato et al. (2014) Archives Biochem. Biophys., 560:83-99. Low-molecular-weight thiol antioxidants useful in the systems, methods, and compositions include glutathione (gamma-glutamylcysteinyl glycine), cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid, and/or dithiothreitol (any of which can also be used in combination with each other at a similar final thiol concentration). ROS scavenging agents useful in the systems, methods, and compositions also include pro-thiols (e.g., L-2-oxothiazolidine-4-carboxylate (OTC)) which are converted to thiols in the cell. In certain embodiments, the plant cell is exposed or treated with enzymatic ROS scavenging agents. Enzymatic ROS scavenging agents include any catalase, ascorbate peroxidase, a dehydroascorbate reductase, guaiacol peroxidase, monodehydroascorbate reductase, a peroxidase, and/or superoxide dismutase. In certain embodiments, an enzymatic ROS scavenging agents is provided in the culture medium. In certain embodiments, an enzymatic ROS scavenging agent or polynucleotides encoding the same can be introduced into the plant cell (e.g., by transient or stable transformation, transfection, or with a delivery agent). A combination of at least one enzymatic and at least one non-enzymatic ROS scavenging agent can also be used. Specific embodiments also include plant cell or plant protoplast cultures wherein the culture medium includes about 20, about 40, or about 60 to about 80, about 100, about 120, or about 150 millimolar Ca 2+ , and/or in which the culture medium includes about 0.1, about 0.25, about 0.5, about 0.75, about 1, or about 2 to about 4, about 6, about 8, or about 10 millimolar low-molecular-weight thiol antioxidant. Further embodiments encompassed are plant cell or plant protoplast cultures wherein the culture medium includes combinations of divalent cations and low-molecular-weight antioxidants, with the individual components present in the culture at concentrations similar to those listed above. In certain embodiments, the plant cells (e.g., plant protoplasts) are exposed to the ROS scavenging agents about 5, 10, 15, 30, or 45 minutes to about 60, 75, 90, or 120 minutes after exposure to the gene-editing molecules and/or S-phase promoting agent. In certain embodiments, the plant cells (e.g., plant protoplasts) are exposed to the ROS scavenging agents prior to or at the same time that they are exposed to the gene-editing molecules and/or S-phase promoting agent. In certain embodiments, the combination of the aforementioned ROS scavenging agents with an S-phase promoting agent provides a synergistic increase in frequencies of gene editing through homology directed repair (HDR) pathways that exceeds the sum of the increases in HDR provided by the ROS scavenging agents and S-phase promoting agents alone. In certain embodiments, the combination of the aforementioned ROS scavenging agents with an S-phase promoting agent and any of the aforementioned divalent cations provides a synergistic increase in frequencies of gene editing through homology directed repair (HDR) pathways that exceeds the sum of the increases in HDR provided by the ROS scavenging agents, S-phase promoting agents, and divalent cations.

In certain embodiments, the plant cell or plant protoplast cultures are exposed to the aforementioned culture media immediately after introduction of a gene editing molecule. In certain embodiments, the plant cell or plant protoplast cultures are exposed to the aforementioned culture media during the time that they are treated with a gene editing molecule and immediately afterwards. In certain embodiments, the plant cell or plant protoplast cultures are exposed to the aforementioned culture media before and/or during the time that they are treated with a gene editing molecule and/or immediately afterwards. Exposure of the plant cell or plant protoplast cultures to the culture media can be for about 1, 2, 4, 6, or 8 to about 12, 18, 24, 36, or 48 hours after introduction of a gene editing molecule. Gene editing molecules can be introduced by methods that include transfection, Agrobacterium -mediated transformation, Agro-infection, electroporation, and the like. In certain embodiments, the plant cell or plant protoplast is maintained at a temperature of about 30Â° C., 32Â° C., 34Â° C., or 36Â° C. to about 38Â° C., 40Â° C., or 42Â° C. for at least about 30, 40, 50, or 60 minutes, or for about 30, 40, 50, 60, to about 70, 80, 90, or 120 minutes, following introduction of the gene editing molecules.

Embodiments of the systems, methods, and compositions provided herein also comprise synthesis phase (S-phase) promoting agents. S-phase promoting agents that can be used include S-phase entry promoting agents, S-phase exit inhibiting agents, S-phase function promoting agents, or any combination of such agents. Non-limiting examples of S-phase entry promoting agents include: agents that inhibit a retinoblastoma protein (pRB) and/or agents that result in increased expression of an E2F transcription factor, including an E2F transcription factor that is deregulated and/or over expressed. Non-limiting examples agents that inhibit a retinoblastoma protein (pRB) include a geminivirus RepA protein, a non-viral protein that binds and inhibits a retinoblastoma protein (pRB) of the plant cell, and/or a cyclin-dependent kinase that phosphorylates pRB as well as polynucleotides encoding any of those proteins or kinases.

Geminivirus RepA proteins and polynucleotides encoding the same that can be used can be obtained either from geminiviruses that infect monocot plants (e.g., maize streak virus (MSV), wheat dwarf virus (WDV)) or from geminiviruses that infect dicot plants (e.g., tobacco yellow dwarf virus, bean yellow dwarf virus (BeYDV), tomato golden mosaic virus (TGMV), Cabbage leaf curl virus (CaLCuV), Sri Lankan cassava mosaic virus (SLCMV), tomato leaf curl virus (ToLCV), beet curly top virus (BCTV), tomato pseudo-curly top virus (TPCTV)). In certain embodiments, the geminivirus RepA protein used in a monocot plant cell is obtained from a geminivirus that infects monocot plants. In certain embodiments, the geminivirus RepA protein used in a dicot plant cell is obtained from a geminivirus that infects dicot plants. In certain embodiments, the geminivirus RepA protein can comprise a conserved Leu-x-Cys-x-Glu (LXCXE; SEQ ID NO: 1) pRb binding motif, where x can be any amino acid. Conserved Leu-x-Cys-x-Glu (LXCXE) pRb binding motifs have been characterized in a variety of geminivirus RepA proteins (Liu et al., Virol. 256(2): 270-279). In certain embodiments, the geminivirus RepA protein can comprise an âhelix 4â pRb binding motif (SEQ ID NO:9). The helix 4 pRb binding motif comprises an 11 amino acid sequence that is highly conserved across all geminivirus genera and that contributes to pRB binding in certain geminiviruses that lack an LXCXE pRB binding domain (Arguello-Astorga, et al. J. Virol. 2004, 78(9): 4817-4826). Useful geminivirus RepA proteins include the proteins set forth in Table 1. In certain embodiments, the geminivirus RepA protein can exhibit at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, a polynucleotide encoding a geminivirus RepA protein that exhibits at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8 is used. In certain embodiments, a geminivirus RepA protein that exhibits at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 3, or 4, and that comprises an LXCXE motif and/or a helix 4 motif is used. In certain embodiments, a geminivirus RepA protein that exhibits at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 6, 7, or 8 and that comprises a helix 4 motif is used. In certain embodiments, a polynucleotide encoding a geminivirus RepA protein that exhibits at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 3, or 4, and that comprises an LXCXE and/or a helix 4 motif is used. In certain embodiments, a polynucleotide encoding a geminivirus RepA protein that exhibits at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 6, 7, or 8 and that comprises a helix 4 motif is used. In certain embodiments, a polynucleotide encoding any of the aforementioned RepA proteins is provided at a rate of about 0.0016, 0.0032, 0.008, 0.016, 0.032, 0.08, or 0.10 to about 0.20, 0.32, 0.8, 1.6, 3.2, 8, or 16 femtomole/cell to cultured plant cells including plant protoplasts in the systems, methods, or compositions disclosed herein. In certain embodiments, the polynucleotide encoding any of the aforementioned RepA proteins is provided to cultured plant cells including plant protoplasts in a system, method, or composition provided herein at a rate of about 0.10 to about 2.0 femtomole/cell.

Geminivirus RepA proteins.

SEQ

Viral

pRb Binding

ID NO

Source

Domain Type (s)

MSV

LXCXE (SEQ ID NO: 1) and

Helix 4 (SEQ ID NO: 9)

WDV

LXCXE (SEQ ID NO: 1) and

Helix 4 (SEQ ID NO: 9)

BeYDV

LXCXE (SEQ ID NO: 1) and

Helix 4 (SEQ ID NO: 9)

TGMV

Helix 4 (SEQ ID NO: 9)

CaLCuV

Helix 4 (SEQ ID NO: 9)

SLCMV

Helix 4 (SEQ ID NO: 9)

ToLCV

Helix 4 (SEQ ID NO: 9)

In certain embodiments, a non-viral protein that binds and inhibits a retinoblastoma protein (pRB) of the plant cell can comprise a conserved Leu-x-Cys-x-Glu (LXCXE) pRb binding motif. A non-viral protein that binds and inhibits a retinoblastoma protein (pRB) of the plant cell includes an Scr protein, orthologues thereof, or variants thereof. Scarecrow (Scr) has been shown to physically bind pRB through a conserved Leu-x-Cys-x-Glu (LXCXE) pRb binding motif located in the Scr protein (Cruz-Ramirez, et al., Cell. 2012 Aug. 31; 150(5): 1002-1015). In certain embodiments, the S-phase promoting agents can thus comprise an exogenous and/or heterologous Scr polypeptide or polynucleotide encoding the same. In certain embodiments, the Scr polypeptide is transiently expressed, overexpressed, and/or provided by inducible expression in the plant cell. In certain embodiments, the S-phase promoting agents can thus comprise a transgene or edited endogenous Scr gene in the plant cell that provides for overexpression and/or inducible expression of Scr. In certain embodiments, the Scr gene is a maize zmSer gene encoding the Ser protein of SEQ ID NO:23 (Lim et al.; Plant Cell. 2000 August; 12(8): 1307-1318), a pea Scr gene (Sassa, et al. Plant Cell Physiol. 2001 April; 42(4):385-94), millet Scr-like gene (Liu, et al., Physiol Mol Biol Plants. 2017 July; 23(3): 629-640; or an Arabidopsis Ser gene encoding the protein of SEQ ID NO:24, or an orthologue of any of such Scr genes. In certain embodiments, a non-viral protein that binds and inhibits a retinoblastoma protein (pRB) of the plant cell can comprise a conserved Leu-x-Cys-x-Glu (LXCXE) pRb binding motif. In certain embodiments, a polynucleotide encoding any of the aforementioned non-viral proteins that binds pRB is provided to cultured plant cells including plant protoplasts in a system, method, or composition provided herein at a rate of about 0.0016, 0.0032, 0.008, 0.016, 0.032, or 0.08 to about 0.32, 0.8, 1.6, 3.2, 8, or 16 femtomole/cell. In certain embodiments, the polynucleotide encoding any of the aforementioned non-viral proteins that binds pRB is provided to cultured plant cells including plant protoplasts in a system, method, or composition provided herein at a rate of about 0.10 to about 2.0 femtomole/cell.

Non-limiting examples of agents that inhibit a plant retinoblastoma protein (pRB) also include CYCD4;2, CYCD6;1, polynucleotides encoding the same, as well as orthologs thereof. CYCD4;2 has been shown to form a kinase complex with CDKA (Kono et al. 2006, Plant Cell Rep. 2006, (6):540-5) whereas a CYCD6;1-CDK complex has been shown to inhibit retinoblastoma protein (pRB) by phosphorylation (Cruz-Ramirez, et al., Cell. 2012 Aug. 31; 150(5): 1002-1015). In certain embodiments, an S-phase promoting agent can thus comprise CYCD4;2 or CYCD6;1 polypeptides and orthologs thereof, polynucleotides encoding those polypeptides or orthologs and optionally a CDKA polypeptide or ortholog thereof or polynucleotide encoding the polypeptide or ortholog. In certain embodiments, the CYCD4;2, CYCD6;1, and/or CDKA polypeptide comprises the polypeptide of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, respectively, or an ortholog thereof.

Systems, methods, and compositions provided herein can also comprise homology-dependent repair (HDR) promoting agents. In certain embodiments, such HDR promoting agents can comprise a protein or a polynucleotide encoding the protein. HDR promoting proteins include CtIP/AtGR1, CYCB1, CDKB1, BRCA1, BRCA2, RAD51, RAD52, RAD54, RPA1, RPA2, RPA3, XRCC3, RECQ4A, MUS81, FANCM, and p53 proteins, and biologically active fragments thereof. Representative examples of amino acid sequences for certain aforementioned HDR promoting proteins include those set forth Table 9 as SEQ ID NO: 316 to 530, 600, or 604, or encoded by SEQ ID NO: 37 to 251, 596, or 602. In certain embodiments, proteins that are orthologous to the proteins include those set forth Table 9 as SEQ ID NO: 316 to 530, 600, or 604 or polynucleotides encoding the same, including SEQ ID NO: 37 to 251, 596, or 602, can be used. Such orthologous proteins will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 316 to 530, 600, or 604. In certain embodiments, any of the aforementioned HDR promoting agents can be provided exogenously. Aforementioned RAD51 proteins, orthologs thereof, biologically active fragments thereof or other RecA type proteins used herein can comprise domains or motifs characteristic of the AAA+superfamily of ATPases that include ATP binding and ATP hydrolysis domains (White and Lauring, Traffic. 2007 December; 8(12): 1657-67) and a helix-hairpin-helix DNA binding motif (Aravind, et al. Nucleic Acid Res. 1999, 27(5): 1223-1242; Prentiss et al. Crit Rev Biochem Mol Biol. 2015; 50(6):453-76). Consensus and example domain and motif sequences that can be found in the aforementioned RAD51 proteins, orthologs thereof, biologically active fragments thereof or other RecA type proteins are set forth in Table 9. Non limiting examples of RAD51 proteins used herein include proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 368-392, or 600, at least one ATP binding domain, at least one ATP hydrolysis domain, and/or at least one helix-hairpin-helix DNA binding motif. In certain embodiments, such RAD51 proteins are encoded by polynucleotides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 89 to 113, or 596. In certain embodiments, any of the aforementioned RAD51 proteins used herein can exhibit ATP binding, single-stranded DNA binding activity in the presence of ATP, DNA dependent ATPase activity, double stranded DNA binding activity, recombinase activity, promote homologous pairing of oligonucleotides, and/or promote DNA strand exchange in homologous recombination (Gupta et al. PNAS 1997, 94 (2) 463-468). In certain embodiments, any of the aforementioned HDR promoting agents used in the systems, compositions, and methods can be from a heterologous source organism. In certain embodiments, any of the aforementioned HDR promoting agents, including an endogenously occurring HDR promoting agent, can be provided in amounts that exceed those found in an untreated control plant cell. In certain embodiments, any of the aforementioned HDR promoting agents, including endogenously occurring HDR promoting agent, can be provided at about 1.5-, 2-, 4-, 5-, 8-, or 10-fold or greater concentrations than found in an untreated control plant cell.

Systems, methods, and compositions provided herein can also comprise non-homologous end-joining (NHEJ) inhibitory agents. In certain embodiments, such NHEJ inhibiting agents can comprise a protein or a polynucleotide encoding the protein. NHEJ inhibiting proteins include CYREN (cell cycle regulator of NHEJ) and i53 (inhibitor of 53BP1) proteins, and biologically active fragments thereof. CYREN and i53 proteins and polynucleotides encoding the same are found in various animals and can thus be obtained from various sources or can be synthetic. Representative examples of amino acid sequences for the aforementioned NHEJ inhibiting proteins include those set forth Table 9 as SEQ ID NO: 599, 603 and 606. In certain embodiments, proteins that are orthologous to the proteins include those set forth Table 9 as SEQ ID NO: 599, 603, and 606 or polynucleotides encoding the same, including SEQ ID NO: 595, 601, 605, and 607, can be used. Such orthologous proteins will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 599, 603, or 606. Examples of orthologous proteins that can be used include CYREN proteins of mammals that include mice, pigs, rabbits, and the like. Aforementioned CYREN proteins, isoforms thereof, orthologs thereof, biologically active fragments thereof can comprise one or more Ku-binding motifs or KBM (Grundy et al. Nat Commun. 2016; 7: 11242). Without seeking to be limited by theory, CYREN proteins, isoforms thereof, orthologs thereof, biologically active fragments thereof comprising the KBM near the N-terminus of those proteins can bind to a Ku70/Ku80 heterodimer or Ku80 and inhibit NHEJ (Arnoult et al. Nature. 2017 Sep. 20; 549(7673): 548-552). Consensus and example KBM that can be found in the aforementioned CYREN proteins, isoforms thereof, orthologs thereof, and biologically active fragments thereof include the R-X-X-P-X-W consensus amino acid sequence, where X is any amino acid (SEQ ID NO: 597) or the sequence RVLPSW (SEQ ID NO: 598). Non limiting examples of CYREN proteins used herein include proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 599 or 603 that comprise at least one KBM, including a KBM of SEQ ID NO: 597 or 598. In certain embodiments, any of the aforementioned CYREN proteins used herein can exhibit binding to a Ku70/Ku80 heterodimer or Ku80. In certain embodiments, any of the aforementioned NHEJ inhibiting agents can be provided exogenously. In certain embodiments, any of the aforementioned NHEJ inhibiting agents used in the systems, compositions, and methods can be from a heterologous source organism. In certain embodiments, any of the aforementioned NHEJ inhibiting agents, including an endogenously occurring NHEJ inhibiting agent, can be provided in amounts that exceed those found in an untreated control plant cell. In certain embodiments, any of the aforementioned NHEJ inhibiting agents, including endogenously occurring NHEJ inhibiting agent, can be provided at about 1.5-, 2-, 4-, 5-, 8-, or 10-fold or greater concentrations than found in an untreated control plant cell.

In certain embodiments, such NHEJ inhibitory agents can comprise a protein or nucleic acid that inhibits expression and/or activity of an endogenous plant cell gene or gene product that promotes NHEJ in the plant cell. In certain embodiments, the plant cell gene or gene product that is targeted for inhibition of expression and/or activity comprises a Ku70 and/or Ku80 (e.g., Ku70/Ku80 protein in Table 9), LigIV, XRCC4, XRCC1, PARP1, PARP2, or PARP3 gene or gene product. Inhibition of expression can be achieved by methods that include introduction of loss-of-function mutations (e.g., by gene editing molecules), by induction or production of RNAi, or by any other method described herein or elsewhere. Representative examples of amino acid sequences encoded by the genes targeted by the aforementioned NHEJ inhibitory agents include those set forth Table 9 as SEQ ID NO: 531 to 594. In certain embodiments, proteins that are orthologous to the proteins include those set forth Table 9 as SEQ ID NO: 531 to 594 or the endogenous plant genes encoding the same can be targeted for such inhibition. Such orthologous proteins will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 531 to 594. In certain embodiments, the endogenous plant cell gene targeted for inhibition of said orthologous protein will comprise or encode a polynucleotide sequence of SEQ ID NO: 252 to 315 or a fragment thereof. In certain embodiments, such fragments will comprise a fragment of SEQ ID NO: 252 to 315 corresponding to an exon of the endogenous plant cell gene that is targeted for inhibition. In certain embodiments, the endogenous plant cell gene targeted for inhibition of said orthologous protein will comprise or encode a polynucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 252 to 315 or a fragment thereof. In certain embodiments, such fragments will comprise a portion of a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a fragment of SEQ ID NO: 252 to 315 corresponding to an exon of the endogenous plant cell gene. In certain embodiments, any of the aforementioned NHEJ inhibitory agents can be provided exogenously. In certain embodiments, any of the aforementioned NHEJ inhibitory agents used in the systems, compositions,

CLAIMS

Claims ( 11 )

What is claimed is:

1. A method for increasing Homology Directed Repair (HDR)-mediated genome modification of a plant cell genome, comprising:

providing genome editing molecules to a plant cell,

wherein the plant cell is exposed for at least 2 hours to at least one of an exogenous, heterologous, and/or overproduced agent comprising a geminivirus RepA protein or biologically active fragment thereof, or polynucleotide encoding said RepA protein or biologically active fragment thereof, or any combination thereof, wherein a geminivirus Rep protein is absent;

wherein the genome editing molecules comprise: (i) an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease, a guide RNA or a polynucleotide encoding a guide RNA, and a donor template polynucleotide or a polynucleotide encoding a donor template polynucleotide; or (ii) a sequence-specific endonuclease and a donor template polynucleotide or a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide;

whereby the genome editing molecules modify the plant cell genome by HDR at a frequency that is increased in comparison to a control.

2. The method of claim 1 , wherein the frequency of HDR is increased by at least 3-fold in comparison to a control method wherein a control plant cell is provided with the genome editing molecules but is not exposed to at least one of an exogenous, heterologous, and/or overproduced plant cell geminivirus RepA protein or biologically active fragment thereof, or polynucleotide encoding said protein or biologically active fragment thereof, or any combination thereof.

3. The method of claim 1 , wherein the plant cell is a monocot plant cell.

4. The method of claim 3 , wherein the monocot plant cell is a barley, maize, millet, oat, rice, rye, sorghum, or wheat plant cell.

5. The method of claim 1 , wherein the plant cell is haploid or diploid.

6. The method of claim 1 , wherein the plant cell is in a culture medium.

7. The method of claim 1 , wherein the culture medium includes about 20 to about 150 mM Ca 2+ .

8. The method of claim 1 , wherein the plant cell is contacted with ascorbic acid, glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, thiocysteine, homocysteine, lipoic acid, dithiothreitol, a tocopherol, a carotenoid, a flavonoid, or combination thereof.

9. The method of claim 1 , wherein the culture medium comprises about 0.1 millimolar to about 10 millimolar glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, thiocysteine, homocysteine, lipoic acid, or dithiothreitol.

10. The method of claim 1 , wherein the culture medium comprises about 20 to about 150 mM Ca 2+ and about 0.1 millimolar to about 10 millimolar glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, thiocysteine, homocysteine, lipoic acid, or dithiothreitol.

11. The method of claim 1 , further comprising the step of isolating and/or growing a plant cell, propagule, or plant obtained from the plant cell comprising the genome modification, wherein the genome of the plant cell, propagule, or plant comprises the genome modification.

US18/179,023

2018-01-22

2023-03-06

Methods for improved plant gene-editing

Active

US12043838B2

( en )

Priority Applications (1)

Application Number

Priority Date

Filing Date

Title

US18/179,023

US12043838B2

( en )

2018-01-22

2023-03-06

Methods for improved plant gene-editing

Applications Claiming Priority (5)

Application Number

Priority Date

Filing Date

Title

US201862620130P

2018-01-22

US201862783301P

2018-12-21

PCT/US2019/014559

WO2019144124A1

( en )

2018-01-22

2019-01-22

Plant gene editing systems, methods, and compositions

US202016963372A

2020-07-20

US18/179,023

US12043838B2

( en )

2018-01-22

2023-03-06

Methods for improved plant gene-editing

Related Parent Applications (2)

Application Number

Title

Priority Date

Filing Date

US16/963,372

Continuation

US11634722B2

( en )

2018-01-22

2019-01-22

Plant gene editing systems, methods, and compositions

PCT/US2019/014559

Continuation

WO2019144124A1

( en )

2018-01-22

2019-01-22

Plant gene editing systems, methods, and compositions

Publications (2)

Publication Number

Publication Date

US20230193303A1

US20230193303A1 ( en )

2023-06-22

US12043838B2

true

US12043838B2 ( en )

2024-07-23

Family

ID=67301221

Family Applications (2)

Application Number

Title

Priority Date

Filing Date

US16/963,372

Active

US11634722B2

( en )

2018-01-22

2019-01-22

Plant gene editing systems, methods, and compositions

US18/179,023

Active

US12043838B2

( en )

2018-01-22

2023-03-06

Methods for improved plant gene-editing

Family Applications Before (1)

Application Number

Title

Priority Date

Filing Date

US16/963,372

Active

US11634722B2

( en )

2018-01-22

2019-01-22

Plant gene editing systems, methods, and compositions

Country Status (3)

Country

Link

( 2 )

US11634722B2

( en )

( 1 )

EP3728577A4

( en )

( 1 )

WO2019144124A1

( en )

Families Citing this family (20)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

BR122021002092B1

( en )

2012-05-28

2022-11-29

Evogene Ltd

METHOD FOR INCREASE GROWTH RATE, BIOMASS, VIGOR, NITROGEN USE EFFICIENCY, AND/OR ABIOTICAL STRESS TOLERANCE, AND/OR REDUCED TIME TO EMERGENCY OF INFLORESCENCE OF A PLANT

US11168319B1

( en )

2017-02-28

2021-11-09

Inari Agriculture Technology, Inc.

Plant cell culture

EP3728577A4

( en )

2018-01-22

2021-12-22

Inari Agriculture, Inc.

PLANT GENE EDITING SYSTEMS, PROCEDURES AND COMPOSITIONS

US11746354B2

( en )

2019-07-19

2023-09-05

Inari Agriculture Technology, Inc.

Homology dependent repair genome editing

EP4021168A4

( en )

2019-08-27

2023-09-13

Relica Genomics Inc.

Transformed plants and methods for making and using the same

WO2021138288A1

( en )

2019-12-31

2021-07-08

Inari Agriculture, Inc.

Delivery of biological molecules to plant cells

WO2021144692A1

( en )

2020-01-14

2021-07-22

Crispr Therapeutics Ag

Methods for increased efficiency of homology-directed repair

EP4114951A4

( en )

2020-03-05

2024-05-08

The Regents Of The University Of California

A method for producing plants with minimized biomass byproduct and associated plants thereof

EP4172340A1

( en )

2020-06-29

2023-05-03

KWS SAAT SE & Co. KGaA

Boosting homology directed repair in plants

WO2022020378A1

( en )

2020-07-20

2022-01-27

Flagship Pioneering, Inc.

Viroid-derived polynucleotides for modifications of plants

US10947552B1

( en )

2020-09-30

2021-03-16

Alpine Roads, Inc.

Recombinant fusion proteins for producing milk proteins in plants

IL301396A

( en )

2020-09-30

2023-05-01

Nobell Foods Inc

Recombinant milk proteins and food compositions containing them

US10894812B1

( en )

2020-09-30

2021-01-19

Alpine Roads, Inc.

Recombinant milk proteins

WO2022094558A1

( en )

2020-10-29

2022-05-05

Pioneer Hi-Bred International, Inc.

Methods and compositions for genome modification

EP4019639A1

( en )

2020-12-22

2022-06-29

KWS SAAT SE & Co. KGaA

Promoting regeneration and transformation in beta vulgaris

UY39997A

( en )

2021-11-01

2023-05-15

Flagship Pioneering Innovations Vii Llc

POLYNUCLEOTIDES FOR MODIFYING ORGANISMS

CN114015666B

( en )

2021-11-09

2022-08-12

å¹¿ä¸çåä¸ç§å¦é¢åä¸çç©åºå ç ç©¶ä¸å¿

Application of OsPARP3 gene in regulation and control of plant drought tolerance

AU2023209447A1

( en )

2022-01-20

2024-07-18

Flagship Pioneering Innovations Vii, Llc

Polynucleotides for modifying organisms

CN121712898A

( en )

2023-05-03

2026-03-20

æè°åä¸åæ°ç¬¬ä¸æéè´£ä»»å¬å¸

Endogenous ribovirus satellite RNA amplification system for plants

WO2025264737A1

( en )

2024-06-20

2025-12-26

Syngenta Crop Protection Ag

High-throughput method for mitochondria dna isolation from plant seeds

Citations (13)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

US5169776A

( en )

1987-06-09

1992-12-08

The United States Of America As Represented By The Secretary Of Agriculture

Method to obtain intact, viable protoplasts from pollen grains

US20040006783A1

( en )

2002-06-13

2004-01-08

The Regents The University Of Califorinia

Compositions and methods for modulating Rop GTPase activity in plants

US8106258B2

( en )

2003-12-23

2012-01-31

Monsanto Technology Llc

Use of a low-oxygen environment in plant transformation

US20150059010A1

( en )

2013-08-22

2015-02-26

Pioneer Hi-Bred International Inc

Genome modification using guide polynucleotide/cas endonuclease systems and methods of use

US20150267189A1

( en )

2012-11-01

2015-09-24

Factor Bioscience Inc.

Methods and products for expressing proteins in cells

WO2016054326A1

( en )

2014-10-01

2016-04-07

The General Hospital Corporation

Methods for increasing efficiency of nuclease-induced homology-directed repair

US20160355838A1

( en )

2013-12-11

2016-12-08

International Rice Research Institute

Anaerobic germination-tolerant plants and related materials and methods

WO2017142923A1

( en )

2016-02-16

2017-08-24

Emendobio Inc.

Compositions and methods for promoting homology directed repair mediated gene editing

WO2018085693A1

( en )

2016-11-04

2018-05-11

Inari Agriculture, Inc.

Novel plant cells, plants, and seeds

US20180223295A1

( en )

2015-05-19

2018-08-09

Kws Saat Se

Methods and hybrids for targeted nucleic acid editing in plants

US20190211344A1

( en )

2016-06-28

2019-07-11

Monsanto Technology Llc

Methods and compositions for use in genome modification in plants

WO2019144124A1

( en )

2018-01-22

2019-07-25

Inari Agriculture, Inc.

Plant gene editing systems, methods, and compositions

US20200080110A1

( en )

2016-12-22

2020-03-12

Keygene N.V.

Method for targeted alteration of duplex dna

2019

2019-01-22

EP19741141.6A

patent/EP3728577A4/en

active

Pending

2019-01-22

US16/963,372

patent/US11634722B2/en

active

Active

2019-01-22

PCT/US2019/014559

patent/WO2019144124A1/en

not_active

Ceased

2023

2023-03-06

US18/179,023

patent/US12043838B2/en

active

Active

Patent Citations (15)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

US5169776A

( en )

1987-06-09

1992-12-08

The United States Of America As Represented By The Secretary Of Agriculture

Method to obtain intact, viable protoplasts from pollen grains

US20040006783A1

( en )

2002-06-13

2004-01-08

The Regents The University Of Califorinia

Compositions and methods for modulating Rop GTPase activity in plants

US8106258B2

( en )

2003-12-23

2012-01-31

Monsanto Technology Llc

Use of a low-oxygen environment in plant transformation

US20150267189A1

( en )

2012-11-01

2015-09-24

Factor Bioscience Inc.

Methods and products for expressing proteins in cells

US20150059010A1

( en )

2013-08-22

2015-02-26

Pioneer Hi-Bred International Inc

Genome modification using guide polynucleotide/cas endonuclease systems and methods of use

US20150082478A1

( en )

2013-08-22

2015-03-19

E I Du Pont De Nemours And Company

Plant genome modification using guide rna/cas endonuclease systems and methods of use

US20160355838A1

( en )

2013-12-11

2016-12-08

International Rice Research Institute

Anaerobic germination-tolerant plants and related materials and methods

WO2016054326A1

( en )

2014-10-01

2016-04-07

The General Hospital Corporation

Methods for increasing efficiency of nuclease-induced homology-directed repair

US20180223295A1

( en )

2015-05-19

2018-08-09

Kws Saat Se

Methods and hybrids for targeted nucleic acid editing in plants

WO2017142923A1

( en )

2016-02-16

2017-08-24

Emendobio Inc.

Compositions and methods for promoting homology directed repair mediated gene editing

US20190211344A1

( en )

2016-06-28

2019-07-11

Monsanto Technology Llc

Methods and compositions for use in genome modification in plants

WO2018085693A1

( en )

2016-11-04

2018-05-11

Inari Agriculture, Inc.

Novel plant cells, plants, and seeds

US20200080110A1

( en )

2016-12-22

2020-03-12

Keygene N.V.

Method for targeted alteration of duplex dna

WO2019144124A1

( en )

2018-01-22

2019-07-25

Inari Agriculture, Inc.

Plant gene editing systems, methods, and compositions

US11634722B2

( en )

2018-01-22

2023-04-25

Inari Agriculture Technology, Inc.

Plant gene editing systems, methods, and compositions

Non-Patent Citations (43)

* Cited by examiner, â Cited by third party

Title

Aravind et al., " Conserved Domains in DNA Repair Proteins and Evolution of Repair Systems ", Nucleic Acids Research, vol. 27, No. 5, pp. 1223-1242, 1999.

Arguello-Astorga et al., " A Novel Motif in Geminivirus Replication Proteins Interacts with the Plant Retinoblastoma-Related Protein ", Journal of Virology, vol. 78, No. 9, pp. 4817-4826, May 2004.

Arnoult et al., " Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN ", Nature, vol. 549, pp. 1-22, 2017.

Baltes et al., " DNA Replicons for Plant Genome Engineering ", The Plant Cell, vol. 26, pp. 151-163, Jan. 2014.

Baxter-Burrell et al., " RopGAP4-dependent Rop GTPase Rheostat Control of Arabidopsis Oxygen Deprivation Tolerance ", Science, vol. 296, No. 5575, pp. 2026-2028, Jun. 14, 2002.

Branco-Price et al., " Genome-Wide Analysis of Transcript Abundance and Translation in Arabidopsis Seedlings Subjected to Oxygen Deprivation ", Annals of Botany, vol. 96, No. 4, pp. 647-666, Aug. 2005.

Chen et al 2014 (adv Tech Biol Med 1:1, p. 1-21 )(Year:2014) 2014.

Cruz-Ramirez et al., " A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division ", Cell, vol. 150, No. 5, pp. 1002-1015, Aug. 2012.

Dan, Yinghui, " Biological functions of antioxidants in plant transformation ", In Vitro Cell Dev. Biol. Plant, vol. 44, pp. 149-161, 2008.

European Patent Office, " Extended European Search Report ", issued in connection to Application No. 19741141.6, 7 pages, mailed Nov. 23, 2021.

Grundy et al., " The Ku-binding Motif is a Conserved Module for Recruitment and Stimulation of Non-Homologous End-Joining Proteins ", Nature Communications, pp. 1-11, 2016.

Gupta et al., " Activities of Human Recombination Protein Rad51 ", Proceedings of the National Academy of Sciences of the U.S.A., vol. 94, pp. 463-468, Jan. 1997.

Gurushidze et al., " Doubled Haploidy as a Tool for Chimaera Dissolutions of TALEN-Induced Mutations in Barley ", Biotechnologies for Plant Mutation Breeding, pp. 129-141, Dec. 2016.

Gurushidze et al., " True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells ", PLOS One, vol. 9, Issue 3, pp. 1-9, Mar. 2014.

Hameed, " Hypoxia up-regulates mitochondrial genome-encoded transcripts in Arabidopsis roots ", Genes Genet Syst, vol. 90, No. 6, pp. 325-334, Mar. 2016.

International Search Report and Written Opinion for PCT/US2019/014559 dated Apr. 15, 2019.

Ishii, " Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process ", Plant Physiol., vol. 88, pp. 26-29, 1988.

Karuppanapandian et al., " Reactive Oxygen Species in Plants: Their Generation, Signal Transduction, and Scavenging Mechanisms ", Austrialian Journal of Crop Science, vol. 5, Issue 6, pp. 709-725, 2011.

Kerpen et al., " Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation ", Frontiers in Plant Science, vol. 10, Issue 56, pp. 1-10, Feb. 2016.

Knight, " Calcium Signaling During Abiotic Stress in Plants ", International Review of Cytology, vol. 195, pp. 269-324, 2000.

Kono et al., " A Distinct Type of Cyclin D, CYCD4;2, Involved in the Activation of Cell Division in Arabidopsis ", Plant Cell Reproduction, vol. 25, pp. 540-545, 2006.

Kushwaha et al., " The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome ", PloS Pathog, vol. 13, No. 8, pp. 1-41, Aug. 2017.

Li et al., " TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice ", Journal of Genetics and Genomics, vol. 43, pp. 297-305, Mar. 2016.

Lim et al., " Molecular Analysis of the SCARECROW Gene in Maize Reveals a Common Basis for Radial Patterning in Diverse Meristems ", The Plant Cell, vol. 12, pp. 1307-1318, Aug. 2000.

Lin et al., " Application of Protoplast Technology to CRISPR/Cas9 Mutagenesis: From Single-Cell Mutation Detection to Mutant Plant Regneration ", Plant Biotechnology, vol. 16, pp. 1295-1310, 2018.

List of Highest Large Cities (https://en.wikipedia.org/wiki/List_of_highest_large_cities).

Liu et al., " Bean Yellow Dwarf Virus RepA, but Not Rep, Binds to Maize Retinoblastoma Protein, and the Virus Tolerates Mutations in the Consensus Binding Motif ", Virology, vol. 256, pp. 270-279, 1999.

Liu et al., " Genome-Wide Identification, Phylogeny and Expression Analyses of SCARECROW-LIKE(SCL) Genes in Millet (Setaria italica) ", Physiology and Molecular Biology of Plants, vol. 23, No. 3, pp. 629-640, 2017.

Oxygen Levels at Altitude (Center for Wilderness Safety https://wildsafe.org/resources/ask-the-experts/altitude-safety-101/ oxygen-levels/).

Papadakis et al., " Reduced Activity of Antioxidant Machinery Is Correlated with Suppression of Totipotency in Plant Protoplasts ", Plant Physiology, vol. 126, pp. 434-444, May 2001.

Pivato et al., " Low-Molecular-Weight Thiols in Plants: Functional and Analytical Implications ", Archives of Biochemistry and Biophysics, vol. 560, pp. 83-99, 2014.

Prentiss et al., " Structure/Function Relationships in RecA Protein-Mediated Homology Recognition and Strand Exchange ", Critical Reviews in Biochemistry and Molecular Biology, vol. 50, No. 6, pp. 453-476, 2015.

Riesenberg et al., " Simultaneous Precise Editing of Multiple Genes in Human Cells ", Nucleic Acids Research, pp. 1-10, 2019.

Roest et al., " Plant Regeneration from Protoplasts: A Literature Review ", Acta. Bot. Neerl., vol. 38, Issue 1, pp. 1-23, Mar. 1989.

Sassa et al., " The Molecular Characterization and in Situ Expression Pattern of Pea SCARECROW Gene ", Plant Cell Physiology, vol. 42, No. 4, pp. 385-394, 2001.

Seybold et al., " CDPK Activation in PRR Signaling ", Methods in Molecular Biology, vol. 1578, pp. 173-183, 2017.

White et al., " AAA + ATPases: Achieving Diversity of Function with Conserved Machinery ", Traffic, vol. 8, pp. 1657-1667, 2007.

White et al., " Calcium in Plants ", Annals of Botany, vol. 92, Issue 4, pp. 487-511, Aug. 2003.

Xie et al., " RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System ", Molecular Plant, vol. 6, pp. 1975-1983, 2013.

Yamauchi et al., " Metallothionein genes encoding ROS scavenging enzymes are down-regulated in the root cortex during inducible aerenchyma formation in rice ", Plant Dignal Behav., vol. 12, No. 11, pp. 1-4, Oct. 2017.

Yongwei et al., " Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement ", Frontiers in Plant Science, vol. 7, Article 1928, pp. 1-14, Dec. 2016.

Yoo et al., " Arabidopsis Mesophyll Protoplasts: A Versatile Cell System for Transient Gene Expression Analysis ", Nature Protocol, vol. 2, Issue 7, pp. 1565-1572, Feb. 2007.

Zhang et al., " Efficient and Transgene-Free Genome Editing in Wheat Through Transient Expression Of CRISPR/Cas9 DNA or RNA ", Nature Communication, 7:12617, Aug. 25, 2016, pp. 1-8.

Also Published As

Publication number

Publication date

US20210363536A1

( en )

2021-11-25

WO2019144124A1

( en )

2019-07-25

US11634722B2

( en )

2023-04-25

US20230193303A1

( en )

2023-06-22

EP3728577A4

( en )

2021-12-22

EP3728577A1

( en )

2020-10-28

Methods for improved plant gene-editing — Inari Agriculture Technology, Inc. (US12043838B2)

Related documents