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WO2025059184A1 - Procédés et compositions pour générer des haploïdes doublés paternels à génome modifié - Google Patents

Procédés et compositions pour générer des haploïdes doublés paternels à génome modifié Download PDF

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Publication number
WO2025059184A1
WO2025059184A1 PCT/US2024/046207 US2024046207W WO2025059184A1 WO 2025059184 A1 WO2025059184 A1 WO 2025059184A1 US 2024046207 W US2024046207 W US 2024046207W WO 2025059184 A1 WO2025059184 A1 WO 2025059184A1
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microspore
plant
cell
polypeptide
plant cell
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PCT/US2024/046207
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English (en)
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Jon Aaron Tucker Reinders
Huaxun YE
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Pioneer Hi-Bred International, Inc.
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Publication of WO2025059184A1 publication Critical patent/WO2025059184A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • sequence listing is submitted electronically as an XML formatted sequence listing with a file named “108431-WO-SEC-l_SequenceListing_ST26” created on September 11, 2024 and having a size of 340 kilobytes and is filed concurrently with the specification.
  • sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • Plant breeding programs identify new cultivars by screening numerous plants to identify individuals with desirable characteristics. Large numbers of progeny from crosses are typically grown and evaluated, ideally across multiple years and environments, to select the plants with the most desirable characteristics.
  • Typical breeding methods cross two parental plants and the filial 1 hybrid (Fi hybrid), is the first filial generation.
  • Hybrid vigor in a commercial Fi hybrid is observed when two parental strains, (typically inbreds), from different heterotic groups are intercrossed.
  • Hybrid vigor the improved or increased function of any biological quality resulting after combining the genetic contributions of its parents, is important to commercial maize seed production and these commercial hybrid performance improvements require the continued development of new inbred parental lines.
  • the one or more polynucleotides encoding the one or more morphogenic developmental genes can comprise a first polynucleotide encoding a WUS polypeptide and a second polynucleotide encoding a BBM polypeptide.
  • the polynucleotide encoding the genetic chromosome doubling factor can comprise a polynucleotide encoding a cyclin D2 polypeptide.
  • the polynucleotide encoding the cyclin D2 polypeptide can be operably linked to an inducible promoter and expression of the cyclin D2 polypeptide can be stimulated when the haploid plant cell is obtained, when the haploid plant cell is cultured, or a combination thereof.
  • expression of the cyclin D2 polypeptide can be induced when the haploid plant cell is cultured.
  • the gene editing component can be a base editing agent comprising a deactivated Cas endonuclease (dCas), for example dCas9 or dCasl2f, complexed to a deaminase and the one or more guide polynucleotides introduce a plurality of nucleobase edits at a plurality of genomic target sites in the haploid plant cell.
  • dCas deactivated Cas endonuclease
  • dCas deactivated Cas endonuclease
  • the diploid plant cell can be from an immature embryo, a 1-5 mm zygotic embryo, or an embryo derived from mature ear-derived seed, a leaf base, a leaf from a mature plant, a leaf tip, an immature inflorescence, non-gametic cells from a tassel, an immature ear, or a silk.
  • regenerating the plant from the diploid plant cell can comprise cultivating a TO transgenic plant comprising the one or more expression constructs.
  • obtaining the haploid plant cell can comprise obtaining a microspore from a tassel of the TO transgenic plant.
  • the haploid plant cell can be a single-cell microspore, a microspore-derived multicellular structure, or a microspore- derived embryo-like structure.
  • the diploid plant cell can be from a 1.5-1.9 mm zygotic embryo.
  • the diploid plant cell can be from a 2.2-2.8 mm zygotic embryo.
  • the diploid plant cell can be from a dicot or a monocot.
  • the diploid plant cell can be a maize diploid cell.
  • the present disclosure also provides methods of generating a haploid plant embryo, the method comprising: (a) providing a plant microspore with an embryogenesis-inducing compound, wherein the embryogenesis-inducing compound is a Polycomb-Repressive Complex 2 (PRC2) inhibitor; (b) obtaining an embryogenic microspore from the plant microspore; and (c) culturing the embryogenic microspore to obtain the haploid plant embryo.
  • PRC2 Polycomb-Repressive Complex 2
  • the present disclosure also provides methods of generating a genome-modified, doubled haploid plant cell, the method comprising: (a) providing to a diploid plant cell: a morphogenic developmental polypeptide; a genetic chromosome doubling polypeptide; a Cas polypeptide; and at least one guide polynucleotide, wherein the Cas polypeptide and the guide polynucleotide form a Cas polypeptide-guide polynucleotide complex capable of binding to and modifying at least one genomic target site in the diploid plant cell; wherein the diploid plant cell is generated from a breeding cross of two parent plants or self-pollination from a parent plant, (b) regenerating a plant from the diploid plant cell resulting in a regenerated plant; (c) obtaining a microspore or microspore-derived structure from the regenerated plant of (b), the microspore comprising a modified genomic target site; (d) culturing the microspore or microspor
  • the Cas polypeptide-guide polynucleotide complex can modify the genomic target site in the diploid plant cell via the Cas polypeptide-guide polynucleotide complex resulting in the modified target site.
  • the microspore or microspore-derived structure comprises the Cas polypeptide and the at least one guide polynucleotide from the regenerated plant and the Cas polypeptide-guide polynucleotide complex modifies the genomic target site in the microspore or microspore-derived structure resulting in the modified target site.
  • the method further comprises providing to the diploid plant cell a polynucleotide of interest that encodes a transgene, wherein the transgene is a selectable marker or a trait of agronomic interest.
  • modifying the genomic target site comprises introducing at least once nucleotide insertion, deletion, substitution, rearrangement, or a combination thereof.
  • the Cas polypeptide can be Cas9 or Casl2f, and the method further comprises providing the diploid plant cell with a donor DNA or a polynucleotide modification template.
  • the Cas polypeptide can be catalytically inactive and complexed to a deaminase to form a base editor.
  • the at least one guide polynucleotide can comprise a plurality of unique guide polynucleotides and the at least one genomic target site can comprise a plurality of unique genomic target sites, and wherein modifying the plurality of genomic target sites comprises introducing a plurality of nucleobase edits at the plurality of genomic target sites via the base editor complexing with each unique guide polynucleotide.
  • the catalytically inactive Cas polypeptide can be dCas9 or dCas!2f
  • the disclosure further provides a method of generating a genome-modified, doubled haploid plant cell, the method comprising: (a) providing to a microspore or microspore-derived structure: a morphogenic developmental polypeptide; a genetic chromosome doubling polypeptide; a Cas polypeptide; and at least one guide polynucleotide, wherein the Cas polypeptide and the guide polynucleotide form a Cas polypeptide-guide polynucleotide complex capable of binding to and modifying at least one genomic target site in the microspore or microspore-derived structure; wherein the microspore or microspore-derived structure is generated from a breeding cross of two parent plants or self-pollination from a parent plant, (b) culturing the microspore or microspore-derived structure to induce embryogenesis and chromosome doubling, wherein chromosome doubling occurs without the use of an exogenous chemical chromosome doubling agent; (c)
  • the method can further comprise providing to the microspore or microspore-derived structure a polynucleotide of interest that encodes a transgene, wherein the transgene is a selectable marker or a trait of agronomic interest.
  • modifying the genomic target site can comprise introducing at least once nucleotide insertion, deletion, substitution, rearrangement, or a combination thereof.
  • the Cas polypeptide can be Cas9 or Casl2f, and the method further comprises providing the microspore or microspore-derived structure with a donor DNA or a polynucleotide modification template.
  • the Cas polypeptide can be catalytically inactive and complexed to a deaminase to form a base editor.
  • the at least one guide polynucleotide can comprise a plurality of unique guide polynucleotides and the at least one genomic target site can comprise a plurality of unique genomic target sites, and wherein modifying the plurality of genomic target sites comprises introducing a plurality of nucleobase edits at the plurality of genomic target sites via the base editor complexing with each unique guide polynucleotide.
  • the catalytically inactive Cas polypeptide can be dCas9 or dCasl2f.
  • the breeding cross can be an Fl biparental cross or a successive filial generation thereof or a backcross or a successive backcross generation thereof.
  • the morphogenic developmental polypeptide, the genetic chromosome polypeptide, and/or the Cas polypeptide can be provided directly as polypeptides.
  • the morphogenic developmental polypeptide, the genetic chromosome polypeptide, and/or the Cas polypeptide can be provided via one or more expression cassettes comprising a polynucleotide sequence encoding the morphogenic developmental polypeptide, a polynucleotide sequence encoding the genetic chromosome polypeptide, and/or a polynucleotide sequence encoding the Cas polypeptide, for example, by bacteria-mediated transformation.
  • the genetic chromosome doubling polypeptide comprises a cyclin delta-2 polypeptide.
  • obtaining the microspore or microspore-derived structure from the regenerated plant can comprise obtaining a microspore from a tassel of the TO transgenic plant.
  • the microspore or microspore-derived structure can be a single-cell microspore, a microspore-derived multicellular structure, or a microspore-derived embryo-like structure.
  • the diploid plant cell can be from a 1.5-1.9 mm zygotic embryo or a 2.2-2.8 mm zygotic embryo
  • the genome-modified, microspore-derived doubled haploid plant cell is from a dicot or a monocot.
  • the doubled haploid plant is from maize.
  • the polynucleotide of interest i.e., transgene
  • the polynucleotide of interest can be randomly inserted into the genome of the diploid plant cell or guided to and inserted at a desired genomic site in the diploid plant cell by the Cas polypeptide-guide polynucleotide complex.
  • the polynucleotide of interest i.e., transgene
  • the polynucleotide of interest can be randomly inserted into the genome of the microspore or microspore-derived structure or guided to and inserted at a desired genomic site in the microspore or microspore-derived structure by the Cas polypeptide-guide polynucleotide complex.
  • the method can further comprise excision of the expression cassette comprising the polynucleotide sequence encoding the morphogenic developmental polypeptide.
  • the expression cassette comprising the polynucleotide sequence encoding the morphogenic developmental polypeptide can be flanked by Cre recombinase loxP sites, and the method further comprises providing to the diploid plant cell Cre recombinase or an expression cassette comprising a polynucleotide sequence encoding Cre recombinase.
  • FIGS. 1 - 6 are flowcharts illustrating various methods of generating a genome-edited doubled haploid plant cell, plantlet, or plant.
  • FIG. 7 is an image of stably transformed regenerated plant from the methods of Example 9.
  • FIG. 8 is an image of embryo-like structures obtained from the methods of Example 9.
  • an “altered target site”, “altered target sequence”, “modified target site”, “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration or modification when compared to a non-altered target sequence.
  • Such alterations or modifications include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).
  • androgenic means induction of androgenesis in which a male gamete is reprogrammed to an embryogenic state and further develops into an embryo or embryo-like structure and plant.
  • the “anther” is part of the stamen containing the microsporangia that is attached to the filament.
  • the microsporangia produce microsporocyte, also known as the microspore mother cell, which then produces four microspores through meiosis. The microspores divide through mitosis to create pollen grains.
  • asexual reproduction means reproduction without the fusion of gametes.
  • the term “before”, in reference to a sequence position refers to an occurrence of one sequence upstream, or 5’, to another sequence.
  • central cell means the female gamete giving rise to the endosperm.
  • coding sequence means the portion of DNA sequence bounded by a start and a stop codon that encodes the amino acids of a protein.
  • crossed or “cross” or “crossing” refers to the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants).
  • progeny i.e., cells, seeds, or plants.
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants).
  • a “deaminase” is an enzyme that catalyzes a deamination reaction. For example, deamination of adenine with an adenine deaminase results in the formation of inosine.
  • Inosine selectively base pairs with cytosine instead of thymine. This results in a post-replicative transition mutation, such that the original A - T base pair transforms into a G - C base pair.
  • cytosine deamination results in the formation of uracil, which can be repaired by cellular repair mechanisms back to a C - T base pair or to a T - A, G - C, or A - T base pair.
  • This heterogeneity in repair can be suppressed by the introduction of a uracil glycosylase inhibitor, such that DNA repair or replication transforms the original C - T base pair into a T - A base pair (Burnett et al. (2022) Frontiers in Genome Editing. 4, 923718).
  • the introduction of a nick promotes the respective base pair change (Burnett et al., 2022).
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
  • Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%.
  • embryogenesis means the formation of an embryo from a sporophytic cell without gamete and seed formation.
  • sporogenesis means the formation of spores from spore mother cells.
  • spore mother cell means the first cell of the reproductive lineage, formed from sporophytic cells in female and male reproductive tissues of the plant.
  • the target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.
  • endogenous target sequence and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell.
  • An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell.
  • transfer cassette means a T-DNA comprising an expression cassette or expression cassettes flanked by the right border and the left border.
  • Cellular reprogramming refers to the method of providing a stimulus to alter the cell fate of a treated cell. Often cellular reprogramming comprises reverting a differentiated, more specialized cell towards an induced pluripotent stem cell state. Such methods can also comprise trans-differentiation, defined as the transformation of a cell other than a stem cell into a second cell type.
  • reprogram or “reprograming” or “reprogrammed” is a process of reverting or sensitizing mature, specialized cells into induced pluripotent stem cells or into cells in an embryonic/embryogenic state capable of being further developed into an embryo or embryolike structure.
  • a population of cells that are being “reprogrammed” not all cells are expected to be “reprogrammed” to the same extent or to the same embryonic/embryogenic state. A mixture or mosaic of cells at various states of reprogramming is generally expected.
  • Methods and compositions provided herein are expected to increase the ratio or percent of cells that are reprogrammed and in a desired embryonic/embryogenic state compared to cells that have not been exposed to the methods and compositions provided herein.
  • Reprograming also refers to the reestablishment of germ cell development.
  • Reprogramming can occur when an embryogenesis inducing agent, such as a polypeptide and/or small molecule compound is contacted with plant cells rendering the plant cells embryogenic.
  • the methods of the present disclosure provide contacting a haploid plant cell with an embryogenesis inducing agent such as for example, a polypeptide and/or a small molecule compound to reprogram cell fate and cause the cell to become embryogenic.
  • an embryogenesis inducing agent such as for example, a polypeptide and/or a small molecule compound to reprogram cell fate and cause the cell to become embryogenic.
  • a polynucleotide encoding an embryogenesis inducing polypeptide may be introduced and expressed in a plant cell wherein the embryogenesis inducing polypeptide impacts surrounding/adjacent cells thereby rendering those surrounding/adjacent cells embryogenic.
  • the cells may be reprogrammed in planta or ex situ.
  • a “cellular reprogramming treatment” is any of the treatments disclosed herein that elicits an embryogenesis response in the contacted cell.
  • a plant cell is treated with a cellular reprogramming treatment inside the tissue of the organism, that is prior to cell isolation or cell extraction for experimentation and/or measurements done in an external environment, it is referred to as an “in planta” treatment or treatment method.
  • Cellular reprogramming affects cell fate and can result in various types of cell fate changes.
  • One cell fate is a cell becoming totipotent, characterized as a cell that can form the sporophyte and extraembryonic cells, such as endosperm cells in the case of plants.
  • Another cell fate is a cell becoming pluripotent, characterized as a cell that can give rise to all the cell types comprising the sporophyte, excluding extraembryonic cells.
  • Embryogenic cells capable of direct organogenesis can be considered as pluripotent.
  • Another cell fate is characterized as a cell becoming multipotent, defined as a cell that can develop into more than one cell type, but being more limited than pluripotent cells, such as plant cells undergoing indirect organogenesis.
  • Reprogramming can also refer to the erasure of epigenetic marks characteristic of a differentiated, or a more specialized cell state and re-establishment of epigenetic marks characteristic of an embryogenic cell state.
  • the disclosure provides a method of generating a genome-edited doubled haploid plant cell, plantlet, or plant, the method comprising (a) providing a diploid plant cell (or a plurality of diploid plant cells) one or more expression constructs expressing one or more morphogenic developmental polypeptides, one or more gene editing components, and a genetic chromosome doubling polypeptide; (b) regenerating a genome-edited plant from the diploid plant cell, wherein the genome-edited plant comprises the one or more expression constructs; (c) obtaining a haploid plant cell from the genome-edited plant; (d) culturing the haploid plant cell to induce, stimulate, or improve embryogenesis and chromosome doubling via one or more culture treatments; and (e) obtaining a genome-edited doubled haploid plant cell, plantlet, or plant.
  • the diploid plant cell is transformed with one or more expression cassettes comprising a polynucleotide encoding a first morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a genetic chromosome doubling polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), and a polynucleotide encoding a site-specific nuclease operably linked to a constitutive promoter.
  • an inducible promoter e.g., a chemically inducible promoter
  • a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter
  • the haploid cell(s) are cultured with one or more treatments to induce, stimulate, or improve embryogenesis and/or chromosome doubling.
  • the haploid cell(s) are treated with the appropriate chemical or chemicals to drive expression of the first and second morphogenic developmental polypeptides.
  • the haploid cell(s) are treated with the appropriate chemical to drive expression of the genetic chromosome doubling polypeptide.
  • the haploid cell(s) can be further treated with exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxy vinylgly cine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • the one or more gene editing components comprises a Cas polypeptide and one or more guide polynucleotides that modify one or more genomic target sites in the diploid plant cell resulting in a genome-edited diploid plant cell that subsequently undergoes regeneration into the genome-edited plant.
  • the one or more guide polynucleotides can be provided to the diploid plant cell as part of the one or more expression constructs (i.e., those expressing the morphogenic developmental polypeptides, the Cas polypeptide, and the genetic chromosome doubling polypeptide).
  • the method comprises regenerating a genome- edited plant from the diploid plant cell by obtaining a TO plant expressing the morphogenic developmental polypeptide(s), the gene editing component(s), and the genetic chromosome doubling polypeptide, selecting tassels from the TO plant, isolating genome-edited microspores, and culturing the microspores as described herein.
  • the disclosure provides a method of generating a genome-edited doubled haploid plant cell, plantlet, or plant, the method comprising (a) providing a diploid plant cell (or a plurality of diploid plant cells) one or more expression constructs expressing one or more morphogenic developmental polypeptides, one or more gene editing components, and a genetic chromosome doubling polypeptide; (b) regenerating a plant from the diploid plant cell, wherein the plant comprises the one or more expression constructs; (c) obtaining a haploid plant cell from the plant, or from a subsequent generation; (d) culturing the haploid plant cell to induce, stimulate, or improve embryogenesis and chromosome doubling via one or more culture treatments and inducing genomic modification of a target site in the haploid plant cell; and (e) obtaining a genome- edited doubled haploid plant cell, plantlet, or plant.
  • the diploid plant cell is transformed with one or more expression cassettes comprising a polynucleotide encoding a first morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a genetic chromosome doubling polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), and a polynucleotide encoding a site-specific nuclease operably linked to an inducible promoter (e.g., a chemically inducible promoter) or a tissue-specific promoter.
  • an inducible promoter e.g., a chemically inducible promoter
  • the haploid cell(s) are cultured with one or more treatments to induce, stimulate, or improve embryogenesis and/or chromosome doubling.
  • the haploid cell(s) are treated with the appropriate chemical or chemicals to drive expression of the first and second morphogenic developmental polypeptides.
  • the haploid cell(s) are treated with the appropriate chemical to drive expression of the genetic chromosome doubling polypeptide.
  • the haploid cell(s) can be further treated with exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxyvinylglycine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • expression of the polynucleotide encoding a site-specific nuclease can be induced via use of an inducible promoter or a tissuespecific, tissue-preferred, or stage-specific promoter.
  • the sitespecific nuclease is a Cas polypeptide
  • the method further comprises providing one or more guide polynucleotides to the haploid cell for genomic modification of the target site.
  • the one or more guide polynucleotides can be provided to the diploid plant cell as part of the one or more expression constructs (i.e., those expressing the morphogenic developmental polypeptides, the Cas polypeptide, and the genetic chromosome doubling polypeptide).
  • the Cas polypeptide and/or the one or more guide polynucleotides can be spatiotemporally expressed, for example, before, during, or after microspore embryogenesis.
  • Methods for spatiotemporal expression control can comprise various known methods, including but not limited to, using a tissue-specific, a tissue-preferred, or a chemically inducible promoter operably linked to the one or more polynucleotides expressing one or more gene editing components.
  • the method comprises regenerating a plant from the diploid plant cell by obtaining a TO plant expressing the morphogenic developmental polypeptide(s), the gene editing component(s), and the genetic chromosome doubling polypeptide, selecting tassels from the TO plant, isolating microspores, and culturing the microspores as described herein.
  • the disclosure provides a method of generating a genome-edited doubled haploid plant cell, plantlet, or plant, the method comprising (a) providing a diploid plant cell (or a plurality of diploid plant cells) one or more expression constructs expressing one or more morphogenic developmental polypeptides and a genetic chromosome doubling polypeptide; (b) regenerating a plant from the diploid plant cell, wherein the plant comprises the one or more expression constructs; (c) obtaining a haploid plant cell from the plant; (d) culturing the haploid plant cell to induce, stimulate, or improve embryogenesis and chromosome doubling via one or more culture treatments; (e) providing the haploid plant cell one or more expression constructs expressing one or more gene editing components to modify a genomic target site in the haploid plant cell; and (f) obtaining a genome-edited doubled haploid plant cell, plantlet, or plant.
  • the diploid plant cell is transformed with one or more expression cassettes comprising a polynucleotide encoding a first morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), and a polynucleotide encoding a genetic chromosome doubling polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter).
  • an inducible promoter e.g., a chemically inducible promoter
  • a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter e.g., a chemically inducible promoter
  • a genetic chromosome doubling polypeptide operably linked to an
  • the haploid cell(s) are cultured with one or more treatments to induce, stimulate, or improve embryogenesis and/or chromosome doubling.
  • the haploid cell(s) are treated with the appropriate chemical or chemicals to drive expression of the first and second morphogenic developmental polypeptides.
  • the polynucleotide encoding the genetic chromosome doubling polypeptide is operably linked to a chemically- inducible promoter, the haploid cell(s) are treated with the appropriate chemical to drive expression of the genetic chromosome doubling polypeptide.
  • the haploid cell(s) can be further treated with exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxy vinylgly cine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • the haploid plant cell can be provided one or more gene editing components to modify a genomic target site in the haploid plant cell.
  • the gene editing components comprise a Cas polypeptide and one or more guide polynucleotides for genomic modification of one or more target sites in the haploid plant cell.
  • the method comprises regenerating a plant from the diploid plant cell by obtaining a TO plant expressing the morphogenic developmental polypeptide(s) and the genetic chromosome doubling polypeptide, selecting tassels from the TO plant, isolating microspores, and culturing the microspores as described herein.
  • tassels can be selected from a subsequent generation plant (e.g., Fl) derived from the TO plant.
  • the disclosure provides a method of generating a genome-edited doubled haploid plant cell, plantlet, or plant, the method comprising (a) providing a diploid plant cell (or a plurality of diploid plant cells) one or more expression constructs expressing one or more morphogenic developmental polypeptides, one or more gene editing components, and a genetic chromosome doubling polypeptide; (b) regenerating a genome-edited plant from the diploid plant cell, wherein the genome-edited plant comprises the one or more expression constructs; (c) inducing, stimulating, or activating the one or more morphogenic developmental polypeptides and the genetic chromosome doubling polypeptide (d) obtaining a haploid plant cell from the genome- edited plant; (e) culturing the haploid plant cell to induce, stimulate, or improve embryogenesis and chromosome doubling via one or more culture treatments; and (f) obtaining a genome-edited doubled haploid
  • the diploid plant cell is transformed with one or more expression cassettes comprising a polynucleotide encoding a first morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a genetic chromosome doubling polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), and a polynucleotide encoding a site-specific nuclease operably linked to a constitutive promoter.
  • an inducible promoter e.g., a chemically inducible promoter
  • a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter
  • the haploid cell(s) can be treated with the appropriate chemical or chemicals to drive expression of the first and second morphogenic developmental polypeptides and/or the genetic chromosome doubling polypeptide.
  • the haploid cells are microspores
  • the intact tassel containing the microspores can be treated with the appropriate chemical or chemicals to drive expression of the first and second morphogenic developmental polypeptides and/or the genetic chromosome doubling polypeptide rather than first removing the microspores from the tassel.
  • the haploid cell(s) can be further treated with exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxyvinylglycine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • the one or more gene editing components comprises a Cas polypeptide and one or more guide polynucleotides that modify one or more genomic target sites in the diploid plant cell resulting in a genome-edited diploid plant cell that subsequently undergoes regeneration into the genome-edited plant.
  • the one or more guide polynucleotide can be provided to the diploid plant cell as part of the one or more expression constructs (i.e., those expressing the morphogenic developmental polypeptides, the Cas polypeptide, and the genetic chromosome doubling polypeptide).
  • the method comprises regenerating a genome- edited plant from the diploid plant cell by obtaining a TO plant expressing the morphogenic developmental polypeptide(s), the gene editing component(s), and the genetic chromosome doubling polypeptide, selecting tassels from the TO plant, isolating genome-edited microspores, and culturing the microspores as described herein.
  • the gene editing components comprise a Cas polypeptide and one or more guide polynucleotides for genomic modification of one or more target sites in the single-cell microspore, the microspore-derived multicellular structure, or the microspore-derived embryo-like structure.
  • the expression construct comprises a polynucleotide sequence encoding a WUS polypeptide, a polynucleotide sequence encoding a BBM/ODP2 polypeptide (or a variant as disclosed herein), a polynucleotide sequence encoding a Cas polypeptide, one or more guide polynucleotides, and a polynucleotide sequence encoding a Zm- CYCD2 polypeptide.
  • One or more of the polynucleotide sequences of the expression construct can be fused with a tRNA-like structure that confers increased stability and/or improved mRNA transport.
  • the disclosure provides a method of generating a haploid plant embryo, the method comprising: (a) providing a single-cell plant microspore, a microspore-derived multicellular structure, or a microspore-derived embryo-like structure with an embryogenesisinducing compound, wherein the embryogenesis inducing compound is a Poly comb -Repressive Complex 2 (PRC2) inhibitor; (b) obtaining an embryogenic microspore from the plant microspore; and (c) culturing the embryogenic microspore to obtain the haploid plant embryo.
  • PRC2 Poly comb -Repressive Complex 2
  • the disclosure provides a method for trait introgression, the method comprising: (a) selecting plants as parents in a breeding cross using their predicted recombination potential using genetic markers (genetic marker is a gene or DNA sequence with a known chromosome location controlling a particular gene or trait), genomic sequence and/or genomic predictions; (b) creating a genetic transformation in one or more of the selected plants utilizing the method of microspore culture transformation described herein to generating a transgenic doubled haploid plant cell, plantlet, or plant as described herein; (c) utilizing any technique resulting in the recovery of a doubled haploid population including, but not limited to embodiments described herein (d) selecting a pre-determined number of microspores, multicellular structures, haploid microspore-derived embryos/ embryo-like-structures, plantlets, or plants with the most advantageous genetic profdes for repeated crossing, where the selection occurs from any number of described entities; (e) crossing the desirable genetic entities and repeating the process until one or multiple markers (genetic marker is a gene or
  • the number of desired entities such as microspores, multi-cellular structures, haploid microspore-derived embryos/embryo-like-structures, plantlets, or plants at each step can be determined using computerized programs. Selection of entities for crossing can be determined using genetic markers, genomic sequence, computerized programs for predicting desired outcomes, or a combination of thereof.
  • the disclosure provides a method of microspore culture transformation, which includes generating transgenic haploid or doubled haploid plant cell, plantlet, or plant, the method comprising: (a) isolating microspores from an Fl breeding cross resultant from a biparental cross, or its corresponding F2 or F3 generations (b) providing the microspores one or more morphogenic developmental polypeptides, a genetic chromosome doubling polypeptide, and one or more transformation cassettes to introduce one or more genes of interest into the microspore, wherein the microspore is a single-cell microspore, a microspore- derived multicellular structure, or a microspore-derived embryo-like structure; (c) culturing the single-cell microspore, the microspore-derived multicellular structure, or the microspore-derived embryo-like structure to induce, stimulate, or improve embryogenesis and chromosome doubling via one or more culture treatments; and (d) obtaining a transformed doubled
  • the single-cell microspore, the microspore-derived multicellular structure, or the microspore-derived embryo-like structure can be further treated with exogenous agents or compounds that induce, stimulate, or improve embryogenesis such as polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxy vinylgly cine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • the transformation cassettes comprise a plasmid DNA containing one or more genes of interest capable of modifying a plant trait in the single-cell microspore, the microspore-derived multicellular structure, or the microspore-derived embryo-like structure, plantlet or plant.
  • One or more morphogenic developmental polypeptides, genetic chromosome doubling polypeptide, and one or more genes of interest can reside in the same plasmid and delivered to the plant cell in a single transformation step, or they can be delivered in multiple plasmids in a single step transformation (co-transformation), or sequentially through multiple transformation steps.
  • the disclosure provides a method of generating a genome-edited haploid plant cell, the method comprising: (a) providing a diploid plant cell (or a plurality of diploid plant cells) one or more expression constructs expressing one or more morphogenic developmental polypeptides, one or more gene editing components, and a genetic chromosome doubling polypeptide; (b) regenerating a genome-edited plant from the diploid plant cell, wherein the genome-edited plant comprises the one or more expression constructs; and (c) obtaining a genome-edited haploid plant cell from the genome-edited plant.
  • the diploid plant cell is transformed with one or more expression cassettes comprising a polynucleotide encoding a first morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), a polynucleotide encoding a genetic chromosome doubling polypeptide operably linked to an inducible promoter (e.g., a chemically inducible promoter), and a polynucleotide encoding a site-specific nuclease operably linked to a constitutive promoter.
  • an inducible promoter e.g., a chemically inducible promoter
  • a polynucleotide encoding a second morphogenic developmental polypeptide operably linked to an inducible promoter
  • the one or more gene editing components comprises a Cas polypeptide and one or more guide polynucleotides that modify one or more genomic target sites in the diploid plant cell resulting in a genome-edited diploid plant cell that subsequently undergoes regeneration into the genome-edited plant.
  • the one or more guide polynucleotides can be provided to the diploid plant cell as part of the one or more expression constructs (i.e., those expressing the morphogenic developmental polypeptides, the Cas polypeptide, and the genetic chromosome doubling polypeptide).
  • the method comprises regenerating a genome-edited plant from the diploid plant cell by obtaining a TO plant expressing the morphogenic developmental polypeptide(s), the gene editing component(s), and the genetic chromosome doubling polypeptide, selecting tassels from the TO plant, and isolating genome- edited microspores, wherein the genome-edited microspores are single-cell microspores, microspore-derived multicellular structures, or microspore-derived embryo-like structures.
  • tassels and the genome-edited single-cell microspores, microspore-derived multicellular structures, or microspore-derived embryo-like structures can be selected from a subsequent generation plant (e.g., Fl) derived from the TO plant.
  • a subsequent generation plant e.g., Fl
  • Bacterial strains useful in the methods of the disclosure include, but are not limited to, a disarmed Agrobacteria, an Ochrobactrum bacteria or a Rhizobiaceae bacteria (U.S. Pat. No. 9,365,859 incorporated herein by reference in its entirety). Standard protocols for particle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev. Biol. - Plant 27: 175-182), Agrobacterium-mQ ⁇ ? Q ⁇ transformation (Jia et al., 2015, Int J. Mol. Sci.
  • Ochrobactrum-mQ a transformation (US2018/0216123 incorporated by reference herein in its entirety) can be used with the methods of the disclosure.
  • Numerous methods for introducing heterologous genes into plants are known and can be used to insert a polynucleotide into a plant host, including biological and physical plant transformation protocols. See, e.g., Miki et al., “Procedure for Introducing Foreign DNA into Plants,” in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993).
  • the methods chosen vary with the host plant and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., (1985) Science 227: 1229-31), Ochrobactrum (US2018/0216123), electroporation, micro-injection and biolistic bombardment.
  • Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of transgenic plants are known and available. See, e.g., Gruber, et al., “Vectors for Plant Transformation,” in Methods in Plant Molecular Biology and Biotechnology, supra, pp. 89-119.
  • Transformation protocols as well as protocols for introducing polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci.
  • each polynucleotide sequence can be delivered in a single expression cassette.
  • the polynucleotide sequences can be delivered in more than expression cassette.
  • morphogenic gene or “morphogenic developmental gene” means a gene that when ectopically expressed stimulates formation of a somatically-derived structure that can produce a plant. More precisely, ectopic expression of the morphogenic gene stimulates the de novo formation of a somatic embryo or an organogenic structure, such as a shoot meristem, that can produce a plant. This stimulated de novo formation occurs either in the cell in which the morphogenic gene is expressed, or in a neighboring cell.
  • a morphogenic gene can be a transcription factor that regulates expression of other genes, or a gene that influences hormone levels in a plant tissue, both of which can stimulate morphogenic changes.
  • a morphogenic gene may be stably incorporated into the genome of a plant or it may be transiently expressed.
  • morphogenic factor means a polynucleotide sequence encoding a morphogenic developmental gene and/or the polypeptide expressed by a morphogenic developmental gene.
  • WUS/WOX genes see US patents 7,348,468 and 7,256,322 and United States Patent Application publications 2017/0121722 and 2007/0271628; Laux et al. (1996) Development 122:87-96; and Mayer etal.
  • Modulation of WUS/WOX is expected to modulate plant and/or plant tissue phenotype including plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, regeneration, somatic embryogenesis initiation, accelerated somatic embryo maturation, initiation and/or development of the apical meristem, initiation and/or development of shoot meristem, initiation and/or development of shoots, or a combination thereof.
  • WUS can induce stem cells in vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J 30:349-359). Also of interest in this regard, would be a MYB1 18 gene (see U.S. Patent 7,148,402), aMYBl 15 gene (see Wang etal. (2008) Cell Research 224-235), a BABYBOOM gene (BBM; see Boutilier et al. (2002) Plant Cell 14: 1737-1749), or a CLAVATA gene (see, for example, U.S. Patent 7,179,963).
  • Morphogenic genes useful in the present disclosure include, but are not limited to, functional WUS/WOX genes.
  • Morphogenic polynucleotide sequences and amino acid sequences of WUS/WOX homeobox polypeptides are useful in the disclosed methods.
  • a “functional WUS/WOX nucleotide” or a “functional WUS/WOX gene” is any polynucleotide encoding a protein that contains a homeobox DNA binding domain, a WUS box, and an EAR repressor domain (Ikeda et al., 2009 Plant Cell 21:3493-3505).
  • the WUSCHEL protein plays a key role in the initiation and maintenance of the apical meristem, which contains a pool of pluripotent stem cells (Endrizzi et al., (1996) Plant Journal 10:967-979; Laux, et al., (1996) Development 122:87-96; and Mayer, et al., (1998) Cell 95:805-815).
  • Arabidopsis plants mutant for the WUS gene contain stem cells that are misspecified and that appear to undergo differentiation.
  • WUS encodes a homeodomain protein which presumably functions as a transcriptional regulator (Mayer, et al., (1998) Cell 95:805-815).
  • the stem cell population of Arabidopsis shoot meristems is believed to be maintained by a regulatory loop between the CLAVATA (CLV) genes which promote organ initiation and the WUS gene which is required for stem cell identity, with the CLV genes repressing WUS at the transcript level, and WUS expression being sufficient to induce meristem cell identity and the expression of the stem cell marker CLV3 (Brand, et al, (2000) Science 289:617-619; Schoof, etal., (2000) Cell 100:635-644).
  • Constitutive expression of WUS in Arabidopsis has been shown to lead to adventitious shoot proliferation from leaves (in planta) (Laux, T., Talk Presented at the XVI International Botanical Congress Meeting, Aug. 1-7, 1999, St. Louis, Mo.).
  • the functional WUS/WOX homeobox polypeptide useful in the methods of the disclosure is a WUS1, WUS2, WUS3, W0X2A, W0X4, W0X5, W0X5A, or W0X9 polypeptide (see, US patents 7,348,468 and 7,256,322 and US Patent Application Publication Numbers 2017/0121722 and 2007/0271628, herein incorporated by reference in their entirety and van der Graaff et al., 2009, Genome Biology 10:248).
  • the functional WUS/WOX homeobox polypeptide useful in the methods of the disclosure can be obtained from or derived from any plant.
  • LEC1 US Patent 6,825,397 incorporated herein by reference in its entirety, Lotan et al., 1998, Cell 93: 1195-1205
  • LEC2 Stone et al., 2008, PNAS 105:3151-3156; Belide et al., 2013, Plant Cell Tiss. Organ Cult 113:543-553
  • KN1/STM Plant Cell Tiss. Organ Cult 113:543-553
  • KN1/STM Tinha et al., 1993. Genes Dev 7:787-795
  • the IPT gene from Agrobacterium Ebinuma and Komamine, 2001, In vitro Cell.
  • Table 1 provides some of the morphogenic developmental genes useful for the methods described herein.
  • a “genetic chromosome doubling factor” refers to a polypeptide, or a polynucleotide encoding the polypeptide, that induces, stimulates, promotes, or improves the doubling of a In haploid chromosome number to 2n (diploid) chromosome number resulting in homozygous chromosomes.
  • a genetic chromosome doubling factor results in diploidization of a haploid plant cell without the use of a chemical chromosome doubling agent, such as colchicine.
  • modulating expression of genes known to impact the plant cell cycle can be used to double the chromosome complement in an embryo.
  • Increasing ploidy level in plant cells can be achieved by modulating expression of genes that stimulate key control points in the cell cycle cell.
  • the genetic chromosome doubling factor is a cell cycle gene such as Cyclin A, Cyclin B, Cyclin C, Cyclin D, Cyclin E, Cyclin F, Cyclin G, and Cyclin H; Pint; E2F; Cdc25; RepA genes and similar plant viral polynucleotides encoding replication-associated proteins. See U.S. Patent Publication No. 2002/0188965 incorporated herein by reference in its entirety.
  • Examples of plant genes whose over-expression stimulates the cell cycle include cyclin-A in tobacco (Yu et al., 2003), cyclin-D in tobacco (Cockcroft et al., 2000, Nature 405:575-79; Thomasger et al., 2002, PNAS 99:6410-6415; Dewitte et al., 2003, Plant Cell 15:79-92)., E2FA in Arabidopsis (De Veylder et al., 2002, EMBO J 21 : 1360-1368), E2FB in Arabidopsis (Magyar et al., 2005, Plant Cell 17:2527-2541).
  • over-expression of viral genes known to modulate plant cell cycle machinery can be used, such as when over-expression of the Wheat Dwarf Virus RepA gene stimulates cell cycle progression (Gl/S transition) and cell division in maize (Gordon- Kamm et al., 2002, PNAS 99:11975-11980).
  • plant genes whose encoded products are known to inhibit the cell cycle have been shown to result in increased cell division when the gene, such as Cyclin-Dependent Kinase Inhibitor (ICK1/KRP), is down -regulated in Arabidopsis (Cheng et al 2013, Plant J 75:642-655).
  • ICK1/KRP Cyclin-Dependent Kinase Inhibitor
  • Methods of down-regulation of a gene such as KRP include expression of an artificial micro-RNA targeted to the KRP mRNA, or expression of a dCas9-repressor fusion that is targeted to the KRP promoter by a gRNA to that sequence.
  • plant genes that are known to specifically impact the process of endoreduplication.
  • ccs52gene or the Dell gene in the methods of the present disclosure, it is expected that over-expression of ccs52 would result in an increased ploidy level as observed in Medicago sativa (Cebolla et al., 1999, EMBO J 18:4476-4484), and that down-regulation of Del l would result in an increased ploidy level as observed in Arabidopsis (Vlieghe et al., 2005, Current Biol 15:59-63). It is expected that other genes that are known to stimulate the cell cycle, the Gl/S transition, or endoreduplication can be used in the methods disclosed herein to increase ploidy level.
  • diploid plant cell(s) are provided a polynucleotide sequence encoding a cyclin D2 polypeptide, such as a maize cyclin D2 polypeptide.
  • a cyclin D2 polypeptide such as a maize cyclin D2 polypeptide.
  • the addition of cyclin D2 overexpression in the formation of a haploid plant cell e.g., a haploid embryo
  • the polynucleotide sequence encoding the maize cyclin D2 polypeptide can comprise a polynucleotide sequence having at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99% sequence identity to SEQ ID NO: 76.
  • the polynucleotide sequence encoding the cyclin D2 polypeptide comprises SEQ ID NO: 76.
  • the cyclin D2 polypeptide can comprise an amino acid sequence having at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99% sequence identity to SEQ ID NO: 77.
  • the cyclin D2 polypeptide comprises SEQ ID NO: 77.
  • the Zm-CYCD2 activity can promote chromosome doubling to a level that exceeds the level associated with spontaneous doubling.
  • a benefit of the methods disclosed herein is that the haploid cell genome doubling provided by Zm- CYCD2 activity occurs in the absence of chemical chromosome doubling agents, such as colchicine.
  • the methods of the present disclosure can increase plant regeneration frequencies of fertile plants. For example, because chemical chromosome doubling agents, such as colchicine can have negative pleiotropic effects that can reduce the number of viable or usable embryoids (see Theor Appl Genet 81 :205-211). Further, using Zm-CYCD2 activity to induce chromosome doubling in microspores reduces the duration and complexity of in vitro tissue culture following microspore isolation as treatment of microspores with an artificial chemical chromosome doubling agent is rendered unnecessary.
  • the genetic chromosome doubling factor can be a truncated Zm-0DP2 polypeptide that promotes chromosome doubling of a haploid plant cell to produce a doubled haploid cell. More specifically, a ZM-ODP2 (TR5) polynucleotide sequence (SEQ ID NO: 79) encoding a Zm-ODP2-(266-669) polypeptide (SEQ ID NO: 80). The Zm-0DP2 (TR5) nucleotide sequence can be operably linked to an inducible and/or tissue-preferred promoter tissue-preferred.
  • the Zm-ODP2-(266-669) polypeptide can be used in the methods described herein in the absence of the Zm-CYCD2 polypeptide (SEQ ID NO: 77).
  • the Zm- ODP2-(266-669) polypeptide can be used in the methods described herein in conjunction with the Zm-CYCD2 polypeptide.
  • the diploid plant cell can be provided one or more expression constructs that express a polynucleotide encoding a first genetic chromosome doubling factor (Zm-CYCD2) and a polynucleotide encoding a second genetic chromosome doubling factor (ZM-ODP2-(266-669).
  • diploid plant cell(s) are provided a polynucleotide comprising the ZM- ODP2 (TR5)-V1 sequence (SEQ ID NO: 82), a PROTEIN LINKER 1 (MODI) sequence (SEQ ID NO: 84), and a transcriptional activator domain sequence, such as AT-CBF1A (MO) (SEQ ID NO: 86) encoding a fusion protein (Zm-ODP2-(266-668):At-CBFla; SEQ ID NO: 89) comprising the ZM-ODP2-(266-668) peptide (SEQ ID NO: 83) fused to a PROTEIN LINKER 1 peptide (SEQ ID NO: 85) fused to a AT-CBF1A transcriptional activator domain (SEQ ID NO: 87).
  • Suitable regulatory domains for fusion with Zm-ODP2-(266-668) include AT-CBF3I (MOI) (SEQ ID NOs: 90 and 91), HHVP-16 (SEQ ID NOs: 92 and 93), and VP16 (SEQ ID NOs: 94 and 95).
  • the methods provided herein can utilize one or more polycomb-repressive complex 2 (PRC2) inhibitors to stimulate, improve, or enhance cellular reprogramming (e.g., embryogenesis) in a haploid plant cell.
  • PRC2 polycomb-repressive complex 2
  • the PRC2 complex contributes to chromatin compaction, and catalyzes the methylation of histone H3 at lysine 27. Tri-methylation at histone 3 lysine 27 (H3K27me3) is associated with transcriptional repression.
  • PRC2 inhibitors suppress PRC2 activity thereby allowing acetylation at histone 3 lysine 27 (H3K27ac) that is associated with increased gene transcription activity.
  • PRC2 inhibitors of the present disclosure include chemical cellular reprogramming agents, such as those shown in Table 2.
  • a “targeted mutation” is a mutation in a gene (referred to as the target gene), including a native gene, made by altering a target sequence within the target gene using any method known to one skilled in the art, including a method involving a guided Cas endonuclease system as disclosed herein.
  • target site refers to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave.
  • the target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.
  • endogenous target sequence and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell.
  • An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell.
  • Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell.
  • Methods for “modifying a target site” and “altering a target site” are used interchangeably herein and refer to methods for producing an altered target site.
  • Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases.
  • CRISPR loci Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs-SPacer Interspersed Direct Repeats constitute a family of recently described DNA loci.
  • CRISPR loci consist of short and highly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to 140 times-also referred to as CRISPR-repeats) which are partially palindromic.
  • the repeated sequences (usually specific to a species) are interspaced by variable sequences of constant length (typically 20 to 58 by depending on the CRISPR locus (W02007/025097 published March 1, 2007).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • a CRISPR locus can include of a CRISPR array, comprising short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.
  • CRISPR-associated CRISPR-associated gene.
  • a Cas polypeptide is further defined as a functional fragment or functional variant of a native Cas polypeptide, or a protein that shares at least 50%, between 50% and 55%, at least 55%, between 55% and 60%, at least 60%, between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100% sequence identity with at least 50, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350
  • Cas endonucleases unwind the DNA duplex at a target sequence and, optionally, cleave at least one DNA strand, as mediated by recognition of the target DNA sequence by a guide polynucleotide (such as, but not limited to, a CRISPR RNA (crRNA) or guide RNA) that is complexed with a Cas endonuclease.
  • a guide polynucleotide such as, but not limited to, a CRISPR RNA (crRNA) or guide RNA
  • crRNA CRISPR RNA
  • Such recognition and cutting of a target DNA sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the target DNA sequence.
  • PAM protospacer-adjacent motif
  • a Cas endonuclease can lack DNA cleavage or nicking activity, but can still specifically bind to a target DNA sequence when complexed with a suitable guide polynucleotide.
  • a suitable guide polynucleotide See also U.S. Patent Application US20150082478 published 19 March 2015 and US20150059010 published 26 February 2015.
  • a “Cas endonuclease” may comprise domains that enable it to function as a double- strand-break-inducing agent.
  • a Cas endonuclease may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas).
  • the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).
  • a “functional fragment”, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a Cas endonuclease are used interchangeably herein, and refer to a portion or subsequence of the Cas endonuclease of the present disclosure in which the ability to recognize, bind to, and optionally unwind, nick or cleave (introduce a single or double-strand break in) the target site is retained.
  • Cas endonucleases that have been described include, but are not limited to, for example: Cas9, Casl2f (Cas-alpha, Casl4), Casl21 (Cas-beta), Casl2a (Cpfl), Casl2b (a C2cl protein), Casl3 (a C2c2 protein), Casl2c (a C2c3 protein), Casl2d, Casl2e, Casl2g, Casl2h, Casl2i, Casl2j, Casl2k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, CaslO, or combinations or complexes of these.
  • the methods and compositions described herein can utilize transposon- associated TnpB, a programmable RNA-guided DNA endonuclease.
  • Cas endonucleases and effector proteins can be used for targeted genome editing (via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas protein or sgRNA.
  • a Cas endonuclease can also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali et al., 2013, Nature Methods Vol. 10: 957-963).
  • a Cas endonuclease, effector protein, or functional fragment thereof, for use in the disclosed methods can be isolated from a native source, or from a recombinant source where the genetically modified host cell is modified to express the nucleic acid sequence encoding the protein.
  • the Cas endonuclease protein can be produced using cell free protein expression systems, or be synthetically produced.
  • Cas endonucleases may be isolated and introduced into a heterologous cell, or may be modified from its native form to exhibit a different type or magnitude of activity than what it would exhibit in its native source. Such modifications include but are not limited to: fragments, variants, substitutions, deletions, and insertions.
  • Fragments and variants of Cas endonucleases can be obtained via methods such as site- directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art such as, but not limiting to, WO2013166113 published 07 November 2013, WO2016186953 published 24 November 2016, and WO2016186946 published 24 November 2016.
  • the Cas endonuclease can comprise a modified form of the Cas polypeptide.
  • the modified form of the Cas polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas protein.
  • the modified form of the Cas protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (US20140068797 published 06 March 2014).
  • the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas” or “deactivated Cas (dCas).”
  • An inactivated Cas/deactivated Cas includes a deactivated Cas endonuclease (dCas).
  • a catalytically inactive Cas endonuclease can be fused to a heterologous sequence to induce or modify activity.
  • a Cas endonuclease can be part of a fusion protein comprising one or more heterologous protein domains (e g., 1, 2, 3, or more domains in addition to the Cas protein).
  • Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA.
  • Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.
  • fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc.).
  • a catalytically inactive Cas can also be fused to a FokI nuclease to generate double-strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).
  • the Cas endonuclease is a fusion protein further comprising a nuclease domain, a transcriptional activator domain, a transcriptional repressor domain, an epigenetic modification domain, a cleavage domain, a nuclear localization signal, a cell-penetrating domain, a translocation domain, a marker, or a transgene that is heterologous to the target polynucleotide sequence or to the cell from which said target polynucleotide sequence is obtained or derived.
  • the nuclease fusion protein comprises Clo51 or Fokl.
  • Cas endonucleases described herein can be expressed and purified by methods known in the art, for example as described in WO/2017/186953.
  • a Cas endonuclease can comprise a heterologous nuclear localization sequence (NLS).
  • a heterologous NLS amino acid sequence herein may be of sufficient strength to drive accumulation of a Cas protein in a detectable amount in the nucleus of a yeast cell herein, for example.
  • the one or more gene editing components comprises a Cas9 endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications in a target polynucleotide sequence.
  • a genome editing system comprises a Cas9 endonuclease, one or more guide polynucleotides, and a donor DNA.
  • Cas9 (formerly referred to as Cas5, Csnl, or Csxl2) is a Cas endonuclease that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence.
  • the canonical Cas9 recognizes a 3’ GC-rich PAM sequence on the target dsDNA, typically comprising an NGG motif.
  • the Cas endonucleases described herein may recognize additional PAM sequences and used to modify target sites with different recognition sequence specificity.
  • a Cas9 polypeptide comprises a RuvC nuclease with an HNH (H-N-H) nuclease adjacent to the RuvC-II domain.
  • the RuvC nuclease and HNH nuclease each can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick).
  • the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al., 2013, Cell 157: 1262-1278).
  • Cas9 endonucleases are typically derived from a type II CRISPR system, which includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component.
  • a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
  • a Cas9 can be in complex with a single guide RNA (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).
  • the type II CRISPR/Cas system from bacteria employs a crRNA and tracrRNA to guide the Cas endonuclease to its DNA target.
  • the crRNA contains the region complementary to one strand of the double strand DNA target and base pairs with the tracrRNA (trans-activating CRISPR RNA) forming a RNA duplex that directs the Cas endonuclease to cleave the DNA target.
  • a guide polynucleotide comprises a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain, and a tracrRNA.
  • a guide polynucleotide comprises a variable targeting domain of 12 to 30 nucleotides and an RNA fragment that interacts with a Cas9 endonuclease.
  • the one or more gene editing components comprises Cas-alpha (e.g., Casl2f) endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications in a target polynucleotide sequence.
  • a genome editing system comprises a Cas-alpha endonuclease, one or more guide polynucleotides, and a donor DNA.
  • a Cas-alpha endonuclease is a functional RNA-guided, PAM-dependent dsDNA cleavage protein of fewer than 800 amino acids, comprising: a C-terminal RuvC catalytic domain split into three subdomains and further comprising bridge-helix and one or more Zinc finger motif(s); and an N-terminal Rec subunit with a helical bundle, WED wedge-like (or “Oligonucleotide Binding Domain”, OBD) domain, and, optionally, a Zinc finger motif.
  • Cas-alpha endonucleases comprise one or more Zinc Finger (ZFN) coordination motif s) that may form a Zinc binding domain. Zinc Finger-like motifs can aid in target and non-target strand separation and loading of the guide polynucleotide into the DNA target. Cas-alpha endonucleases comprising one or more Zinc Finger motifs can provide additional stability to a ribonucleoprotein complex on a target polynucleotide. Cas-alpha endonucleases comprise C4 or C3H zinc binding domains.
  • a Cas-alpha endonuclease can function as a double-strand-break-inducing agent, a singlestrand-break inducing agent, or as a nickase.
  • a catalytically inactive Cas-alpha endonuclease can be used to target or recruit to a target DNA sequence but not induce cleavage.
  • a catalytically inactive Cas-alpha protein can be combined with a base editing molecule, such as a cytidine deaminase or an adenine deaminase.
  • the one or more gene editing components comprises a Cas endonuclease, one or more guide polynucleotides, and optionally donor DNA
  • editing a target polynucleotide sequence comprises nonhomologous end-joining (NHEJ) or homologous directed repair (HDR) following a Cas endonuclease-mediated doublestrand break.
  • NHEJ nonhomologous end-joining
  • HDR homologous directed repair
  • chromosomes The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell 14: 1121- 31; Pacher et al., (2007) Genetics 175:21-9).
  • the double-strand break can be repaired by homologous recombination between homologous DNA sequences.
  • gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152: 1173-81).
  • the one or more gene editing components comprises a Cas endonuclease, one or more guide polynucleotides, and a donor DNA.
  • donor DNA is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. Once a double-strand break is introduced in the target site by the endonuclease, the first and second regions of homology of the donor DNA can undergo homologous recombination with their corresponding genomic regions of homology resulting in exchange of DNA between the donor and the target genome.
  • the provided methods result in the integration of the polynucleotide of interest of the donor DNA into the double-strand break in the target site in the plant genome, thereby altering the original target site and producing an altered genomic target site.
  • HDR and/or NHEJ can be used to modify a genomic (DNA) target site.
  • Such alterations or modifications include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).
  • Genomic modifications via a Cas polypeptide- guide polynucleotide complex include SDN-1. SDN-2, and SDN-3 mutations of a genomic target site. Genomic modification from the methods described herein also includes chromosomal rearrangement.
  • the one or more gene editing components comprises a base editing agent and a plurality of guide polynucleotides and editing a target polynucleotide sequence comprises introducing a plurality of nucleobase edits in the target polynucleotide sequence resulting in a variant nucleotide sequence.
  • One or more nucleobases of a target polynucleotide can be chemically altered, in some cases to change the base from one type to another, for example from a Cytosine to a Thymine, or an Adenine to a Guanine.
  • a plurality of bases for example 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more 90 or more, 100 or more, or even greater than 100, 200 or more, up to thousands of bases may be modified or altered, to produce a plant with a plurality of modified bases.
  • Any base editing complex such as a base editing agent associated with an RNA-guided protein, may be used to target and bind to a desired locus in the genome of an organism and chemically modify one or more components of a target polynucleotide.
  • Site-specific base conversions can be achieved to engineer one or more nucleotide changes to create one or more edits into the genome.
  • These include for example, a site-specific base edit mediated by an C»G to T*A or an A»T to G»C base editing deaminase enzymes (Gaudelli et al., Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.” Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al.
  • Base editors convert C->T (or G->A on the opposite strand) or an adenine base editor that would convert adenine to inosine, resulting in an A->G change within an editing window specified by the gRNA.
  • Any molecule that effects a change in a nucleobase is a “base editing agent” or a “base editor”.
  • the creation of single double-strand breaks and the subsequent repair via HDR or NHEJ is not ideal for quantitative traits.
  • An observed phenotype includes both genotype effects and environmental effects.
  • the genotype effects further comprise additive effects, dominance effects, and epistatic effects.
  • the probability of no effect per any single edit can be greater than zero, and any single phenotypic effect can be small, depending on the method used and site selected.
  • Double-stranded break repair can additionally be “noisy” and have low repeatability.
  • a multiplexed approach to base editing in an organism has the potential to create a plurality of significant phenotypic variations in one or a few generations, with a positive directional bias to the effects.
  • the organism is a plant.
  • a plant or a population of plants with a plurality of edits can be cross-bred to produce progeny plants, some of which will comprise multiple pluralities of edits from the parental lines. In this way, accelerated breeding of desired traits can be accomplished in parallel in one or a few generations, replacing time-consuming traditional sequential crossing and breeding across multiple generations.
  • a base editing deaminase such as a cytidine deaminase or an adenine deaminase, may be fused to an RNA-guided endonuclease that can be deactivated (“dCas”, such as a deactivated Cas9 or Casl2f) or partially active (“nCas”, such as a Cas9 nickase or Casl2f nickase) so that it does not cleave a target site to which it is guided.
  • dCas such as a deactivated Cas9 or Casl2f
  • nCas such as a Cas9 nickase or Casl2f nickase
  • the dCas forms a functional complex with a guide polynucleotide that shares homology with a polynucleotide sequence at the target site, and is further complexed with the deaminase molecule.
  • the guided Cas endonuclease recognizes and binds to a double-stranded target sequence, opening the double-strand to expose individual bases.
  • the deaminase deaminates the cytosine base and creates a uracil.
  • Uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C.
  • DNA replication or repair mechanisms then convert the Uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly G in the original G-C pair) to an Adenine, creating a T-A pair.
  • U to T thymine
  • Adenine a thymine
  • DNA replication or repair mechanisms then convert the Uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly G in the original G-C pair) to an Adenine, creating a T-A pair.
  • the one or more gene editing components comprises a prime editing agent and a guide polynucleotide and editing a target nucleotide sequence comprises introducing one or more insertions, deletions, or nucleobase swaps in a target nucleotide sequence without generating a double-stranded DNA break.
  • the prime editing agent is a Cas polypeptide fused to a reverse transcriptase, wherein the Cas polypeptide is modified to nick DNA rather than generating doublestrand break.
  • This Cas-polypeptide-reverse transcriptase fusion can also be referred to as a “prime editor” or “PE”.
  • the guide polynucleotide comprises a prime editing guide polynucleotide (pegRNA), and is larger than standard sgRNAs commonly used for CRISPR gene editing (e.g., >100 nucleobases).
  • the pegRNA comprises a primer binding sequence (PBS) and a template containing the desired or target RNA sequence at its 3’ end.
  • the PE:pegRNA complex binds to a target DNA sequence and the modified Cas polypeptide nicks one target DNA strand resulting in a flap.
  • the PBS on the pegRNA binds to the DNA flap and the target RNA sequence is reverse transcribed using the reverse transcriptase.
  • the edited strand is incorporated into the target DNA at the end of the nicked flap, and the target DNA sequence is repaired with the new reverse transcribed DNA.
  • Genome modification via a Cas polypeptide may be used to effect a genotypic and/or phenotypic change in the haploid or diploid plant cell.
  • a change is preferably related to an improved trait of interest or an agronomically-important characteristic, the correction of an endogenous defect, or the expression of some type of expression marker.
  • the trait of interest or agronomically-important characteristic is related to the overall health, fitness, or fertility of the plant, the yield of a plant product, the ecological fitness of the plant, or the environmental stability of the plant.
  • the trait of interest or agronomically- important characteristic is selected from the group consisting of: agronomics, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial product production.
  • the trait of interest or agronomically-important characteristic is selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered starch content, altered carbohydrate content, altered sugar content, altered fiber content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant not comprising a modification derived from the methods or compositions herein.
  • traits of interest to confer an agronomically-important characteristic can be provided at the diploid or haploid cell stage as transgenes.
  • a transgenic trait can be provided to a diploid or haploid cell, with such a modification being distinct from a genomic modification by a Cas polypeptide- guide polynucleotide complex.
  • a diploid or haploid cell can be provided with both a Cas polypeptide, one or more guide polynucleotides, and a polynucleotide sequence encoding a transgenic trait.
  • the methods described herein utilize microspores around the first pollen mitosis (i.e., late uninucleate to early binucleate stage) for embryogenesis induction.
  • Isolation of microspores typically occurs after a tassel pretreatment in a reduced temperature environment to improve the androgenic response.
  • a commonly used technique is to place foil wrapped tassels at 10°C for between 1 to 21 days, preferably for 5-14 days.
  • preculture of anthers in a mannitol solution for example 0.3M liquid mannitol plus 50 mg/L ascorbic acid, can be practiced (US Patent 5,322,789 and US Patent 5,445,961 incorporated herein by reference in their entireties).
  • tassels Prior to use, tassels can be surface-sterilized in a 40% Clorox (8.25% Sodium Hypochlorite diluted v/v) solution with two drops of Tween 80 for approximately fifteen minutes, with gentle agitation on a reciprocal shaker. The tassels are then rinsed three or more times in sterile water at room temperature and placed in a large petri dish and typically left uncovered for 1-1.5 hours under aseptic conditions to allow any excess water to evaporate prior to microspore isolation.
  • Another method of surface sterilization includes placing spikelets detached from the tassel into permeable baskets that are then submerged in a 40% Clorox (8.25% Sodium Hypochlorite diluted v/v) solution with two drops of Tween 80 for fifteen minutes followed by rinsing as described above.
  • Clorox 8.25% Sodium Hypochlorite diluted v/v
  • the spikelets are placed in a large petri dish and typically left uncovered for 1-1.5 hours to allow excess water to evaporate prior to microspore isolation.
  • Isolation procedures for maize anthers and spikelets include, but not limited to, glass rod maceration methods (Pescitelli, et al., (1990) Plant Cell Rep. 8:628-31), blending methods, razor blade tissue cutting methods (see US Patent 5,445,961 incorporated herein by reference in its entirety), tissue homogenizer methods (Gaillard, etal., (1991) Plant Cell Rep. 10:55-8), and tissue grinder methods (Mandaron etal., (1990) Theor Appl Genet 80: 134-138).
  • microspores Following isolation of microspores from the surrounding somatic tissue, the microspores are typically immediately separated from any anther debris and placed into fresh isolation medium. Numerous media compositions are known in the art. A common method of separating microspores from anther debris is to pass a blended microspore anther debris slurry from the isolation procedure through a sieve (Pescitelli (1989) Plant Cell Rep. 7:673-6, Gaillard, et al., (1991), and US Patent 5,445,961 incorporated herein by reference in its entirety). Alternatively, the microspore anther debris slurry is passed through several layers of cheesecloth or a mesh filter (Coumans, (1989) Plant Cell Rep. 7:618-21).
  • Further separation can be performed using a discontinuous density centrifugation method or additional filtration methods, including but not limited, to methods using a sucrose or Percoll gradient (Coumans, (1989), Pescitelli et al., (1990)).
  • selection of cells captured at the 20-30% interface of a Percoll gradient ranging from 20-50% after centrifugation at 225g for 3 min can be further separated using a final, high sucrose (0.44M) centrifugation method (Gaillard, et al., (1991)).
  • Further variations to separation methods are known in the art (Vergne etal., (1991) In: Negrutiu I. (ed) BioMethods. Birkhauser, Basel, Boston, Bedinger and Edgerton, (1990) Plant Physiol. 92:474-9, Gaillard, et al., (1991)) and can be optimized as needed.
  • Specific media used during isolation typically consists of 6% sucrose, 50 mg/L acorbic acid, 400 mg/L proline, 0.05 mg/L biotin and 10 mg/L nicotinic acid (see Petolino and Genovesi (1994) The Maize Handbook, Freeling, M., Walbot, V. (eds) Springer- Verlag, New York).
  • Various other media and solutions used for the culturing of maize microspores are similar to those used for other cereal tissue culture procedures and various modifications can be used (see Genovesi and Magill, (1982) Plant Cell Rep. 1:257-60, Martin and Widholm, (1996) Plant Cell Rep.
  • Common features for maize culture media typically include the use of N6, NLN, or YP basal salt formulations with relatively high sugar concentrations (6-12%) that may have constituents including triiodobenzoic acid, various phytohormones, and/or proline.
  • microspores can be cultured in a vessel, such as a sterile petri dish, in a 9% sucrose induction medium.
  • a vessel such as a sterile petri dish
  • the cultured cells can be incubated at 28°C under dark conditions.
  • the methods provided herein can utilize one or more treatments to promote, improve, or increase microspore embryogenesis.
  • microspores can be cultured in the presence of polycomb repressive complex 2 (PRC2) inhibitors and/or ethylene inhibitors.
  • PRC2 polycomb repressive complex 2
  • ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxyvinylglycine) and ethylene signal perception inhibitors (e.g., silver nitrate).
  • Microspores can also be contacted with small molecule kinase inhibitors that promote cellular reprogramming from an initial haploid gametic cell fate to an embryogenic cell fate, including, N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, anthra(l,9-cd)pyrazol-6(2H)-one, 4-(4-Fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4- pyridyl)lH-imidazole, and N-benzyl-2-(pyrimidin-4-ylamino)-l,3-thiazole-4-carboxamide (see US11447786B2; incorporated by reference).
  • small kinase inhibitors that promote cellular reprogramming from an initial haploid gametic cell fate to an embryogenic cell fate, including, N-[(2
  • the polynucleotides expressing the first morphogenic developmental polypeptide, the second morphogenic developmental polypeptide, and/or the genetic chromosome doubling factor utilize sulfonylurea-mediated control of gene expression have been previously disclosed (see US20110287936A1; incorporated by reference).
  • microspores are staged, extracted, isolated, and cultured in vitro in the presence of a sulfonylurea compound, such as ethametsulfuron, to regulate expression of the WUS, BBM, and Zm-CYCD2 expression constructs.
  • regenerating a genome-edited plant from the diploid plant cell or “regenerating a plant from the diploid plant cell” comprises obtaining a TO plant expressing the aforementioned expression cassette(s) from the transformed plant cell, fertilizing the TO plant with donor pollen, obtaining T1 progeny, and optionally identifying a genome-edited plant from the T1 progeny.
  • “regenerating a genome-edited plant from the diploid plant cell” or “regenerating a plant from the diploid plant cell” comprises obtaining a TO plant expressing the aforementioned expression cassette(s) from the transformed plant cell, isolating tassels from the TO plant, and optionally identifying genome- edited tassels.
  • Reporter genes or selectable markers can be included in the expression constructs described herein.
  • suitable reporter genes known in the art can be found in, for example, Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al., (1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) Current Biology 6:325-330, herein incorporated by reference in their entirety.
  • Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance or resistance to herbicides.
  • suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron, et al., (1985) Plant Mol. Biol.
  • the expression construct(s) expressing the one or more morphogenic developmental polypeptides, the one or more gene editing components, and/or the genetic chromosome doubling polypeptide can optionally further comprise a reporter gene useful for monitoring the presence or absence of the T-DNA.
  • the expression construct(s) expressing the one or more morphogenic developmental polypeptides, the one or more gene editing components, and/or the genetic chromosome doubling polypeptide can optionally further comprise a reporter gene useful for detecting cellular reprogramming.
  • the expression construct can comprise a fluorescent reporter gene operably linked to an embryogenic promoter, such as but not limited to, the HvLTP promoter.
  • regulatory element refers to a nucleic acid molecule having gene regulatory activity, i.e. one that has the ability to affect the transcriptional and/or translational expression pattern of an operably linked transcribable polynucleotide.
  • gene regulatory activity thus refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and/or translation of that operably linked transcribable polynucleotide molecule.
  • Gene regulatory activity may be positive and/or negative and the effect may be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive qualities as well as by quantitative or qualitative indications.
  • Cis regulatory elements are regulatory elements that affect gene expression. Cis regulatory elements are regions of non-coding DNA that regulate the transcription of neighboring genes, often as DNA sequences in the vicinity of the genes that they regulate. Cis regulatory elements typically regulate gene transcription by encoding DNA sequences conferring transcription factor binding.
  • promoter is an exemplary regulatory element and generally refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence comprises proximal and more distal upstream elements, the latter elements are often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • Promoters may be derived in their entirety from a native gene or may be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different regulatory elements may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • a “plant promoter” is a promoter capable of initiating transcription in plant cells.
  • Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobium, which comprise genes expressed in plant cells. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids or sclerenchyma. Such promoters are referred to as “tissue-preferred” promoters. Promoters that initiate transcription only in certain tissues are referred to as “tissue-specific”.
  • a “cell-type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • An “inducible” or “regulatable” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light.
  • Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development.
  • Tissue-preferred, cell-type specific, developmentally-regulated and inducible promoters are members of the class of “non-constitutive” promoters.
  • a “constitutive” promoter is a promoter that causes a nucleic acid fragment to be expressed in most cell types at most times under most environmental conditions and states of development or cell differentiation.
  • a “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect numerous parameters including, processing of the primary transcript to mRNA, mRNA stability and/or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225 236).
  • Promoters useful in the methods of the present disclosure include those that modulate paternal embryogenesis. For paternal embryogenesis, exemplary promoters include tasselpreferred promoters, anther-preferred promoters, and tapetum-preferred promoters.
  • Tissuespecific, tissue-preferred, or stage-specific regulatory elements further include the anther-specific LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), microspore-specific promoters such as the apg gene promoter (Twell, et al., (1993) Sex. Plant Reprod. 6:217-224), tapetum-specific promoters such as the TA29 gene promoter (Mariani, et al., (1990) Nature 347:737; U.S. Pat. No.
  • stamen-specific promoters such as the MS26 gene promoter, MS44 gene promoter, MS45 gene promoter, the 5126 gene promoter, the BS7 gene promoter, the PG47 gene promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No. 5,545,546; Zheng et al., (1993) Plant J 3(2):261-271), the SGB6 gene promoter (U.S. Pat. No. 5,470,359), G9 gene promoter (U.S. Pat. No. 5,8937,850; U.S. Pat. No. 5,589,610), the SB200 gene promoter (WO 2002/26789), and the like.
  • a tissue-preferred promoter active in cells of male reproductive organs is particularly useful in the methods of the present disclosure.
  • the polynucleotides expressing the morphogenic developmental polypeptides, gene editing components, and/or genetic chromosome doubling polypeptides can be operably linked to a suitable promoter.
  • Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters.
  • An “inducible” or “repressible” promoter can be a promoter which is under either environmental or exogenous control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, or the presence of light.
  • exogenous control of an inducible or repressible promoter can be affected by providing a suitable chemical or other agent that via interaction with target polypeptides result in induction or repression of the promoter.
  • Inducible promoters also encompass constitutive promoters modified with a chemically inducible operator sequence, such as a chemically inducible operator sequence within the UBI-ZM or CaMV 35S promoter.
  • Inducible promoters include heat-inducible promoters, estradiol-responsive promoters, chemically-inducible promoters, and the like.
  • Pathogen inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.
  • PR proteins pathogenesis-related proteins
  • PR proteins PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al. (1992) The Plant Cell 4: 645- 656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116.
  • Inducible promoters useful in the present methods include GLB1, OLE, LTP2, HSP17.7, HSP26, 15 HSP18A, and XVE promoters.
  • a chemically-inducible promoter can be repressed by the tetracycline repressor (TETR), the ethametsulfuron repressor (ESR), or the chlorsulfuron repressor (CSR), and de-repression occurs upon addition of tetracycline-related or sulfonylurea ligands.
  • the repressor can be TETR and the tetracycline-related ligand is doxycycline or anhydrotetracycline. 20 (Gatz, C., Frohberg, C. and Wendenburg, R.
  • the repressor can be ESR and the sulfonylurea ligand is ethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl, chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron, sulfosulfuron, trifloxysulfuron, 25 foramsulfuron, iodosulfuron, prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron (US20110287936 incorporated herein by reference in its entirety).
  • the CSR ligand is chlorsulfuron.
  • a “constitutive” promoter is a promoter which is active under most conditions. Promoters useful in the present disclosure include those disclosed in WO2017/112006 and those disclosed in US Provisional Application 62/562,663. Constitutive promoters for use in expression of genes in plants are known in the art. Such promoters include but are not limited to 35S promoter of cauliflower mosaic virus (CaMV 35S) (Depicker et al. (1982) Mol. Appl. Genet. 1 :561-573; Odell et al.
  • CaMV 35S cauliflower mosaic virus
  • constitutive promoters useful in the methods of the present disclosure include UBI, 5 LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZMH1B PRO (1.2 KB), IN2-2, NOS, the -135 version of 35S, and ZM-ADF PRO (ALT2) promoters.
  • Promoters useful in the present disclosure include those disclosed in U.S. Pub. No. US20170121722, U.S. Pub. No. US20180371480 and U.S. Pub. No. 8,710,206 (incorporated by reference herein in their entireties).
  • the polynucleotides encoding the one or more morphogenic developmental polypeptides can be operably linked to an inducible promoter such that expression of the morphogenic developmental polypeptide(s) is stimulated or induced during haploid collection or haploid culture.
  • Expression of the genetic chromosome doubling polypeptide can be similarly regulated.
  • the polynucleotide encoding the one or more gene editing components can be operably linked to a constitutive promoter, resulting in genomic modification of a diploid plant cell provided with the polynucleotide and a guide polynucleotide.
  • initiation of gene-editing can be stimulated or induced at the haploid cell stage (e.g., during haploid cell culture) using an inducible promoter, a tissue-specific promoter, or a tissue-preferred promoter.
  • the expression construct can comprise a polynucleotide sequence encoding a recombinase operably linked to an embryogenic promoter, such as but not limited to, the HvLTP promoter.
  • the disclosed methods can be used to introduce into plants polynucleotides useful for the targeted integration of nucleotide sequences into a plant.
  • the disclosed methods can be used to introduce T-DNA expression cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site.
  • the target site contains at least a set of non-identical recombination sites corresponding to those on the T-DNA expression cassette.
  • the exchange of the nucleotide sequences flanked by the recombination sites is affected by a recombinase.
  • the disclosed methods can be used for the introduction of T-DNA expression cassettes for targeted integration of nucleotide sequences, wherein the T-DNA expression cassettes which are flanked by non- identical recombination sites recognized by a recombinase that recognizes and implements recombination at the nonidentical recombination sites. Accordingly, the disclosed methods and composition can be used to improve efficiency and speed of development of plants containing non- identical recombination sites.
  • the disclosed methods can further comprise methods for the directional, targeted integration of exogenous nucleotides into a transformed plant.
  • the disclosed methods use novel recombination sites in a gene targeting system which facilitates directional targeting of desired genes and nucleotide sequences into corresponding recombination sites previously introduced into the target plant genome.
  • a nucleotide sequence flanked by two non-identical recombination sites is introduced into one or more cells of an explant derived from the target organism's genome establishing a target site for insertion of nucleotide sequences of interest.
  • a second construct, or nucleotide sequence of interest, flanked by corresponding recombination sites as those flanking the target site is introduced into the stably transformed plant or tissues in the presence of a recombinase protein. This process results in exchange of the nucleotide sequences between the non-identical recombination sites of the target site and the T-DNA expression cassette.
  • the transformed plant prepared in this manner may comprise multiple target sites; i.e., sets of non-identical recombination sites. In this manner, multiple manipulations of the target site in the transformed plant are available.
  • target site in the transformed plant is intended a DNA sequence that has been inserted into the transformed plant's genome and comprises non-identical recombination sites.
  • Examples of recombination sites for use in the disclosed method are known.
  • the two- micron plasmid found in most naturally occurring strains of Saccharomyces cerevisiae encodes a site-specific recombinase that promotes an inversion of the DNA between two inverted repeats. This inversion plays a central role in plasmid copy-number amplification.
  • FLP protein catalyzes site-specific recombination events.
  • the minimal recombination site has been defined and contains two inverted 13 -base pair (bp) repeats surrounding an asymmetric 8- bp spacer.
  • the FLP protein cleaves the site at the junctions of the repeats and the spacer and is covalently linked to the DNA via a 3'phosphate.
  • Site specific recombinases like FLP cleave and religate DNA at specific target sequences, resulting in a precisely defined recombination between two identical sites. To function, the system needs the recombination sites and the recombinase. No auxiliary factors are needed.
  • the yeast FLPVFRT site specific recombination system has been shown to function in plants. To date, the system has been utilized for excision of unwanted DNA. See, Lyznik et at. (1993) Nucleic Acid Res. 21 : 969-975.
  • the present disclosure utilizes non-identical FRTs for the exchange, targeting, arrangement, insertion and control of expression of nucleotide sequences in the plant genome.
  • a transformed organism of interest such as an explant from a plant, containing a target site integrated into its genome is needed.
  • the target site is characterized by being flanked by non-identical recombination sites.
  • a targeting cassette is additionally required containing a nucleotide sequence flanked by corresponding non-identical recombination sites as those sites contained in the target site of the transformed organism.
  • a recombinase which recognizes the non- identical recombination sites and catalyzes site-specific recombination is required.
  • the recombinase can be provided by any means known in the art. That is, it can be provided in the organism or plant cell by transforming the organism with an expression cassette capable of expressing the recombinase in the organism, by transient expression, or by providing messenger RNA (mRNA) for the recombinase or the recombinase protein.
  • mRNA messenger RNA
  • flanking recombination sites it is intended that the flanking recombination sites are not identical in sequence and will not recombine or recombination between the sites will be minimal. That is, one flanking recombination site may be a FRT site where the second recombination site may be a mutated FRT site.
  • the non-identical recombination sites used in the methods of the present disclosure prevent or greatly suppress recombination between the two flanking recombination sites and excision of the nucleotide sequence contained therein.
  • any suitable non-identical recombination sites may be utilized in the present disclosure, including FRT and mutant FRT sites, FRT and lox sites, lox and mutant lox sites, as well as other recombination sites known in the art.
  • suitable non-identical recombination site implies that in the presence of active recombinase, excision of sequences between two non-identical recombination sites occurs, if at all, with an efficiency considerably lower than the recombinationally-mediated exchange targeting arrangement of nucleotide sequences into the plant genome.
  • suitable non-identical sites for use in the present disclosure include those sites where the efficiency of recombination between the sites is low; for example, where the efficiency is less than about 30 to about 50%, preferably less than about 10 to about 30%, more preferably less than about 5 to about 10 %.
  • the recombination sites in the targeting cassette correspond to those in the target site of the transformed plant. That is, if the target site of the transformed plant contains flanking non-identical recombination sites of FRT and a mutant FRT, the targeting cassette will contain the same FRT and mutant FRT non-identical recombination sites.
  • the recombinase which is used in the disclosed methods, will depend upon the recombination sites in the target site of the transformed plant and the targeting cassette. That is, if FRT sites are utilized, the FLP recombinase will be needed. In the same manner, where lox sites are utilized, the Cre recombinase is required. If the non-identical recombination sites comprise both a FRT and a lox site, both the FLP and Cre recombinase will be required in the plant cell.
  • the FLP recombinase is a protein which catalyzes a site-specific reaction that is involved in amplifying the copy number of the two-micron plasmid of S. cerevisiae during DNA replication. FLP protein has been cloned and expressed. See, for example, Cox (1993) Proc. Natl. Acad. Sci. U. S. A. 80: 4223-4227.
  • the FLP recombinase for use in the present disclosure may be that derived from the genus Saccharomyces. It may be preferable to synthesize the recombinase using plant preferred codons for optimum expression in a plant of interest. See, for example, U. S. Application Serial No. 08/972,258 filed November 18, 1997, entitled “Novel Nucleic Acid Sequence Encoding FLP Recombinase,” herein incorporated by reference.
  • the bacteriophage recombinase Cre catalyzes site-specific recombination between two lox sites.
  • the Cre recombinase is known in the art. See, for example, Guo et al. (1997) Nature 389: 40-46; Abremski et al. (1984) J. Biol. Chem. 259: 1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet. 22: 477-488; and Shaikh et al. (1977) J. Biol. Chem. 272: 5695-5702. All of which are herein incorporated by reference. Such Cre sequence may also be synthesized using plant preferred codons.
  • a T-DNA comprises LOXP sites positioned so that the one or more expression constructs described herein can be excised by recombinase activity, resulting in the final expression cassettes encoding a first selectable marker, such as a phosphomannose isomerase (PMI) expression cassette, operably linked to a constitutive UBI promoter and an herbicide or antibiotic resistance marker, such as a highly resistant acetolactate synthase (HRA), expression cassette operably linked to a constitutive UBI promoter.
  • a first selectable marker such as a phosphomannose isomerase (PMI) expression cassette
  • PMI phosphomannose isomerase
  • HRA highly resistant acetolactate synthase
  • This T-DNA design allows for the conditional activity of the expression cassettes within the two LOXP sites and it is expected this conditional activity can be removed via recombinase activity being provided to a microspore-derived cell.
  • Recombinase activity can be provided using various methods known in the art, including but not limited to, providing to a microspore-derived plant cell the activity of a recombinase, such as Cre, using a recombinase polypeptide or a polynucleotide sequence encoding a recombinase polypeptide.
  • the expression con struct/ s) expressing the one or more morphogenic developmental polypeptides, the one or more gene editing components, and/or the genetic chromosome doubling polypeptide can optionally further comprise a polynucleotide sequence encoding a recombinase, for example a Cre recombinase, operably linked to an inducible promoter, a tissue-specific promoter, or a tissue-preferred promoter.
  • a recombinase expression the one or more morphogenic developmental polypeptides, the one or more gene editing components, and/or the genetic chromosome doubling polypeptide can be excised following embryogenesis stimulation or induction in the haploid plant cell.
  • a breeding cross In agriculture involving plants, a breeding cross generally refers to the process of mating two genotypically distinct plants to produce offspring with desired traits from both parents. This technique is fundamental in plant breeding, including corn (maize) breeding, to enhance crop performance, yield, and resistance to diseases and pests. There are several types of breeding crosses, including single-cross, double-cross, and backcross, each with specific applications depending on the breeding objectives.
  • Inbred Lines An early-stage step in maize breeding involves creating inbred lines. These are lines that have been self-pollinated for several generations (or developed through a double- haploid (DH) process) to produce substantially genetically uniform plants. Each inbred line has specific desirable traits.
  • Single-Cross Hybrids A single-cross hybrid is produced by crossing two inbred lines. The resulting hybrid, known as an F 1 hybrid, exhibits hybrid vigor or heterosis, which means it is more vigorous and productive than either parent. Generally, all plants of the same single-cross hybrid are genetically identical. At every locus where the two inbred parents possess different alleles, the single-cross hybrid (i.e, Fl) is heterozygous. For example, crossing inbred lines B73 and Mol7 produces a hybrid that is taller and has larger ears than either parent.
  • Double-Cross Hybrids This involves crossing two single-cross hybrids; that is crossing the progeny generated from two single-cross hybrids. The resulting offspring combine traits from four different inbred lines, which can further enhance hybrid vigor and adaptability.
  • B ackcrossing This method involves crossing a hybrid with one of its parental inbred lines to introduce or reinforce specific traits. For example, if a hybrid has a desirable trait from one parent but lacks disease resistance from the other, backcrossing can help incorporate the resistance trait while retaining the hybrid’s other advantages.
  • Recurrent Selection This method is used to improve populations of open-pollinated varieties. It involves selecting the best individuals from a population, intercrossing them, and then selecting the best from the progeny over multiple generations.
  • the methods described herein can be used for transformation of diploid cells from a broad range of plant species including, but no limited to, monocots and dicots.
  • monocot plants that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), wheat (Triticum species, for example Triticum aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa s
  • Examples of dicot plants include, but are not limited to, soybean (Glycine max), Brassica species (for example but not limited to: oilseed rape or Canola) (Brassica napus, B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum).
  • soybean Glycine max
  • Brassica species for example but not limited to: oilseed rape or Canola
  • Brassica napus for example but not limited to: oilseed rape or Canola
  • Brassica napus for example but not limited to: oilseed rape or Canola
  • Brassica napus for example but
  • Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals, and conifers.
  • safflower Carthamus tinctorius
  • sweet potato Ipomoe
  • Vegetables that can be used include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Locopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • the diploid plant cell(s) are maize plant cells.
  • Various maize-derived explants can be used, including immature embryos, 1-5 mm zygotic embryos, and embryos derived from mature ear-derived seed, leaf bases (the portion of the leaf immediately proximal to its attachment point to the petiole or stem), leaves from mature plants, leaf tips, immature inflorescences, tassel, immature ear, and silks.
  • the methods described herein utilize 1.5-1.9 mm maize zygotic embryos or 2.2-2.8 mm maize zygotic embryos.
  • the present disclosure also includes plants obtained by any of the disclosed methods herein.
  • the present disclosure also includes seeds from a plant obtained by any of the methods or compositions disclosed herein.
  • plant refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny of the same.
  • plant refers to whole plants, plant organs (e g., leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny of the same.
  • Plant cells can be differentiated or undifferentiated (e.g.
  • callus undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf cells, root cells, phloem cells and pollen).
  • Plant cells include, without limitation, cells from seeds, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristem, somatic meristems, organogenic callus, protoplasts, embryos derived from mature ear- derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassel, immature ear, silks, cotyledons, immature cotyledons, meristematic regions, callus tissue, cells from leaves, cells from stems, cells from roots, cells from shoots, gametophytes, sporophytes, pollen, microspores, multicellular structures (MCS), and embryo-like structures (ELS).
  • MCS multicellular structures
  • ELS embryo-like structures
  • Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells in culture (e. g., single cells, protoplasts, embryos, and callus tissue).
  • the plant tissue may be in a plant or in a plant organ, tissue, or cell culture. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.
  • Progeny, variants and mutants of the regenerated plants are also included within the scope of the disclosure, provided these progeny, variants and mutants are derived from regenerated plants made using the methods and compositions disclosed herein and/or comprise the introduced polynucleotides disclosed herein.
  • a fertile plant is a plant that produces viable male and female gametes and is self-fertile. Such a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material comprised therein.
  • Other embodiments of the disclosure can involve the use of a plant that is not self-fertile because the plant does not produce male gametes, or female gametes, or both, that are viable or otherwise capable of fertilization.
  • the present disclosure finds use in the breeding of plants comprising one or more edited alleles created by the methods disclosed herein.
  • the edited alleles influence the phenotypic expression of one or more traits, such as plant health, growth, or yield.
  • two plants may be crossed via sexual reproduction to create progeny plant(s) that comprise some or all of the edits from both parental plants.
  • Agrobacterium tumefaciens harboring a binary donor vector was streaked out from a -80°C frozen aliquot onto solid 12R medium and cultured at 28°C in the dark for 2-3 days to make a master plate.
  • a single colony or multiple colonies of Agrobacterium were picked from the master plate and streaked onto a second plate containing 81 OK medium and incubated at 28°C in the dark overnight.
  • Agrobacterium infection medium 700A; 5 ml
  • 100 mM 3'-5'-Dimethoxy-4'- hydroxyacetophenone acetosyringone; 5 pL
  • acetosyringone 5 pL
  • the suspension (1 ml) was transferred to a spectrophotometer tube and the optical density (550 nm) of the suspension was adjusted to a reading of about 0.35-1.0.
  • the Agrobacterium concentration was approximately 0.5 to 2.0 x 109 cfu/mL.
  • the final Agrobacterium suspension was aliquoted into 2 mL microcentrifuge tubes, each containing about 1 mL of the suspension. The suspensions were then used as soon as possible.
  • Agrobacterium can be prepared for transformation by growing in liquid medium.
  • a 125-ml flask is prepared with 30 ml of 557A medium (10.5 g/1 potassium phosphate dibasic, 4.5 g/1 potassium phosphate monobasic anhydrous, 1 g/1 ammonium sulfate, 0.5 g/1 sodium citrate dehydrate, 10 g/1 sucrose, 1 mM magnesium sulfate) and 30 pL spectinomycin (50 mg/mL) and 30 pL acetosyringone (20 mg/mL).
  • a half loopful of Agrobacterium from a second plate is suspended into the flasks and placed on an orbital shaker set at 200 rpm and incubated at 28°C overnight.
  • the Agrobacterium culture is centrifuged at 5000 rpm for 10 min.
  • the supernatant is removed and the Agrobacterium infection medium (700A) with acetosyringone solution is added.
  • the bacteria is resuspended by vortex and the optical density (550 nm) of the Agrobacterium suspension is adjusted to a reading of about 0.35 to 2.0.
  • Ears of a maize (Zea mays L.) cultivar were surface-sterilized for 15-20 min in 20% (v/v) bleach (5.25% sodium hypochlorite) plus 1 drop of Tween 20 followed by 3 washes in sterile water.
  • Immature embryos (IES) were isolated from ears and were placed in 2 ml of the Agrobacterium infection medium (700A) with acetosyringone solution. The optimal size of the embryos varies based on the inbred, but for transformation with WUS2 and ZM-ODP2 a wide size range of immature embryo sizes was used.
  • the Agrobacterium infection medium (81 OK) was drawn off and 1 ml of the Agrobacterium suspension was added to the embryos and the tube was vortexed for 5-10 sec. The microfuge tube was incubated for 5 min in the hood. The suspension of Agrobacterium and embryos were poured onto 7101 (or 562V) co-cultivation medium. Any embryos left in the tube were transferred to the plate using a sterile spatula. The Agrobacterium suspension was then drawn off and the embryos placed axis side down on the media. The plate was incubated in the dark at 21 °C for 1-3 days of co-cultivation and embryos were then transferred to resting medium (605B medium) without selection.
  • resting medium (605B medium
  • microspores Prior to selecting tassels for microspore culture, microspores were staged to an appropriate stage, between the uninucleate to binucleate stage. Tassels with anthers and microspores at the appropriate stage were detached. Each tassel was individually wrapped in aluminum foil and incubated at 10°C for 1-21 days.
  • tassels Prior to microspore isolation, tassels were surface sterilized with a 30% v/v ChloroxTM solution and rinsed with sterile water. Anthers and spikelets were isolated using methods known in the art. Microspores were then separated from anther debris and placed in fresh isolation medium.
  • microspores are cultured in a sterile petri dish in a 9% sucrose induction medium. Microspores are first incubated at a temperature above or below 28°C for a period of time and then incubated at 28°C under dark conditions.
  • microspores are treated with one or more Polycomb-repressive complex 2 (PRC2) inhibitors as shown in Table 4. Untreated microspores will serve as a control.
  • the dosage of the PRC2 inhibitor(s) and the duration of microspore exposure to the PRC2 inhibitor treatment will vary based on the cultivar.
  • Example 6 Method for Obtaining a Paternally-derived Doubled Haploid Plant Using a Genetic Chromosome Doubling Factor
  • Plasmid PHP94809 (SEQ ID NO: 74), designed to activate microspore embryogenesis, contained a T-DNA having a polynucleotide encoding i.) a WUSCHEL expression cassette operably linked to a chemically inducible NOS promoter and ii.) a BABY BOOM expression cassette operably linked to a chemically inducible UBI promoter.
  • Plasmid PHP94810 (SEQ ID NO: 75), designed to provide simultaneous activation of microspore embryogenesis and in vivo chromosome doubling within a microspore, contained a T-DNA having a polynucleotide encoding i.) a WUSCHEL expression cassette operably linked to a chemically inducible NOS promoter, ii.) a BABY BOOM expression cassette operably linked to a chemically inducible UBI promoter, iii.) a ZM-CYCD2 expression cassette operably linked to a chemically inducible CaMV promoter.
  • TO transgenic plants hemizygous for plasmid PHP94809 or PHP94810 were obtained from the population of transformed immature diploid embryos and single copy TO plants were identified and grown to maturity. Each TO plant having a donor ear was fertilized with wild-type pollen to produce T1 generation seed. T1 progeny were grown, DNA was isolated from leaf tissues, and the presence/absence of the T-DNA was determined. T1 progeny were scored and “null” progeny lacking inheritance of a T-DNA were isolated separately from “hemizygous” progeny that were scored as having a single copy T-DNA insertion. [0352] 6B. ZM-CYCD2 transcript levels in response to chemical induction treatments
  • microspores were staged, extracted, isolated, and cultured in vitro in the presence of a sulfonylurea compound, ethametsulfuron, to regulate expression of the WUS, BBM, and Zm-CYCD2 expression cassettes encoded in plasmid PHP94810.
  • a sulfonylurea compound ethametsulfuron
  • Microspore samples were cultured at 28°C under dark conditions and sampled after three and six days of incubation. The samples were frozen using liquid nitrogen and RNA was isolated using known methods. WUS, BBM, and Zm-CYCD2 transcript levels were measured relative to the constitutive level of a tubulin gene using real-time quantitative PCR. The average relative fold gene expression change demonstrated that these three expression cassettes were up-regulated in response to ethametsulfuron-mediated induction (see Table 5), thus demonstrating the chemical induction method worked as expected.
  • Table 5 WUS, BBM, and Zm-CYCD2 transcript levels following ethametsulfuron-mediated induction
  • Ploidy analysis using flow cytometry was performed on microspore-derived cells obtained from two independent PHP94810 events.
  • the microspore-derived cell samples were embryolike structures obtained from 20-day old cell cultures treated with ethametsulfuron (0.1 mg/L) and cultured as described above.
  • the ploidy analysis results showed 64% (11 of 17 samples) and 55% (5 of 9 samples) exhibited diploid (2n) cytometry patterns for the first and second PHP94810 events, respectively.
  • Zm-CYCD2 promoted haploid chromosome doubling, and that this chromosome doubling effect was achieved in the absence of chemical chromosome doubling agents, such as colchicine.
  • the level of chromosome doubling in response to ZM-CYCD2 exceeded the level associated with spontaneous chromosome doubling in maize (0-21.4%).
  • Plants were regenerated from microspore-derived embryoids. Regeneration was improved in response to ZM-CYCD2 activity (PHP94810) as compared to control (lacking ZM-CYCD2 activity; PHP94809) with an average of 2.6 versus 2.0 regenerated progeny per donor plant, respectively. Following regeneration, the plants were transplanted to soil for growth to maturity using methods known in the art. It is anticipated the regenerated, mature plants will have improved fertility in response to ZM-CYCD2 activity.
  • Example 7 Method for Obtaining a Genome-edited Paternally-derived Doubled Haploid Plant
  • the following experiment demonstrates providing simultaneous microspore embryogenesis induction, chromosome doubling, and gene editing activity to a maize haploid cell in vivo to obtain a genome-edited, paternally-derived doubled haploid plant.
  • Plasmid RV053111 contains a T-DNA having a polynucleotide encoding i.) a WUSCHEL expression cassette operably linked to a chemically inducible NOS promoter, ii.) a BABY BOOM/ZM-Ovule Developmental Protein 2 (BBM/ZM-ODP2) expression cassette operably linked to a chemically inducible UBI promoter, iii.) a ZM-CYCD2 expression cassette operably linked to a chemically inducible CaMV promoter, iv) a CRISPR-Cas expression cassette operably linked to a constitutive UBI promoter, v.) two gRNA expression cassettes designed for CRISPR-Cas recruitment to the NAC7 locus (SEQ ID NO: 81), and vi.) a repressor protein,
  • BBM/ZM-ODP2 BABY BOOM/ZM-Ovule Developmental Protein 2
  • TO transgenic plants hemizygous for plasmid RV053111 are obtained from the population of transformed immature maize embryos and single copy TO plants identified and grown to maturity. Each TO plant having a donor ear was fertilized with wild-type pollen to produce T1 generation seed. T1 progeny were grown, DNA was isolated from leaf tissues, and the presence/absence of the T-DNA was determined. Microspores are staged, extracted, isolated, and cultured in vitro. [0361 ] It is anticipated that Cas/gRNA-mediated editing of the maize genome can provide NAC7 edited alleles of microspore-derived plant cells. DNA isolated from the microspore-derived plant cells is used to evaluate the site directed mutation.
  • Example 8 Method for Obtaining a Genome-edited Paternally-derived Doubled Haploid Plant in vitro
  • the following experiment demonstrates gene-editing a microspore-derived maize cell obtained from an initially transformation-incompetent haploid tissue when used with the methods described in Examples 5 and 6. This method transforms a wild-type, microspore-derived cell using a mixture of Agrobacterium strains.
  • a first Agrobacterium strain comprising plasmid RV006010 (SEQ ID NO: 96) is mixed with a second Agrobacterium strain comprising plasmid RV020636 (SEQ ID NO: 97).
  • the Agrobacterium mixture contains a ratio of 95% of the Agrobacterium comprising plasmid RV006010 to 5% of the Agrobacterium comprising plasmid RV020636.
  • the Agrobacterium mixture is used to transform a paternally-derived plant cell.
  • a first plant cell is transformed with plasmid RV020636 expressing a WUS2 polypeptide, which can act in a cell non-autonomous manner to activate somatic embryogenesis in a second plant cell lacking a T-DNA provided by plasmid RV020636.
  • the second plant cell is transformed with a T-DNA provided by plasmid RV006010.
  • the second plant cell is contacted by the WUS2 polypeptide from the first plant cell, resulting in de novo somatic embryogenesis in the second plant cell, wherein the somatic embryo comprises a genome-edited target site. It is expected that the somatic embryo can be regenerated into a plant, the plant being regenerated from a microspore- derived cell.
  • Plasmid RV006010 provides Cre activity to enable excision of the T-DNA polynucleotide sequence encoding the CAS protein, two gRNAs (for multiplex or base editing of multiple genomic target sites), and the Cre recombinase, resulting in a T-DNA conferring a cyan color marker and kanamycin resistance. It is anticipated the regenerated genome-edited, paternally- derived plant will only retain a single LOXP site, commonly referred to as a “LOXP scar” site. It is further anticipated the method enables modifying a maize genome using a microspore-derived cell obtained from an initially transformation-incompetent haploid tissue.
  • Plasmid PHP109457 contained a T-DNA polynucleotide with an expression cassette encoding a Casl2fl polypeptide operably linked to Zea mays ubiquitin regulatory elements (5’ Zm-UBI promoter, Zm-UBI 5’UTR, Zm-UBI intron 1; 3’ Zm-UBI terminator). Additional expression cassettes comprised a polynucleotide encoding two guide RNA, ZM-TARGET-GENE-X-CR12 and ZM-TARGET-GENE-X-CR13, which complex with and recruit Casl2fl to the locus encoding a TARGET-GENE-X polypeptide.
  • a third expression cassette comprised a polynucleotide encoding a Zea mays optimized neomycin phosphotransferase-B gene product operably linked to a Z/w-UBI promoter, Z/M-5’ UTR, Zm-UBI intron 1 and S. bicolor UBI terminator.
  • the Agrobacterium mixture contained a ratio of 80% of the Agrobacterium comprising plasmid PHP109457 to 20% of the Agrobacterium comprising plasmid RV020636.
  • Agrobacterium infection was performed by adding 1 mL of the Agrobacterium mixture containing acetosyringone (200 pM final concentration) to the macroscopic structures that were first vortexed for 5 seconds and then incubated at room temperature for 5 minutes under sterile conditions. The Agrobacterium mixture was removed after 5 min. and each macroscopic structure was transferred to a petri dish containing co-cultivation media with further removal of any excess liquid of the Agrobacterium mixture. Co-cultivation was performed for 48 hours at 21°C under dark conditions. After co-cultivation, all infected tissue was transferred to resting media for 24 hours.
  • haploid chromosome doubling can be combined with haploid chromosome doubling to improve fertility restoration.
  • a chemical chromosome doubling agent such as colchicine can be provided to the transformed haploid cells before, during, or after plantlet regeneration.
  • haploid chromosome doubling can be performed using a haploid genetic chromosome doubling factor, such as ZM-CYCD2.
  • Example 9 A Co-transformation Method for Obtaining a Genome-edited Paternally- derived Doubled Haploid Plant
  • This method transforms a wild-type, diploid cell using a mixture of three Agrobacterium strains, wherein two of the three T-DNA are stably integrated with one T-DNA providing paternal trait activities and a second T-DNA providing a Cas polypeptide and one or more guide RNAs.
  • This co-transformation method enables providing genome editing components separately from the paternal trait activities, thereby offering flexibility to change the Cas polypeptide, guide RNAs, or both.
  • This method further enables producing paternally-derived doubled haploid plants having a plurality of genome modifications.
  • This co-transformation method used a first Agrobacterium strain comprising plasmid PHP88158 (SEQ ID: 100), a second Agrobacterium strain comprising plasmid PHP94810 (SEQ ID: 75), and a third Agrobacterium strain comprising plasmid PHP110677 (SEQ ID: 98).
  • the Agrobacterium mixture contains a ratio of 10% of the Agrobacterium comprising plasmid PHP88158 to 45% of the Agrobacterium comprising plasmid PHP94810 and 45% of the Agrobacterium comprising plasmid PHP110677.
  • the Agrobacterium mixture was used to transform a diploid plant cell.
  • a first plant cell was transformed with plasmid PHP88158 expressing a WUS2 polypeptide, which acted in a cell non-autonomous manner to activate somatic embryogenesis in a second plant cell lacking a T-DNA provided by plasmid PHP88158.
  • the second plant cell was transformed with two independent T-DNA strands provided by plasmids PHP94810 and PHP110677.
  • the second plant cell was contacted by the WUS2 polypeptide from the first plant cell, resulting in de novo somatic embryogenesis in the second plant cell, wherein the somatic embryo comprised a gene-edited target site. It is expected that the somatic embryo can be regenerated into a plant, the plant being regenerated from a diploid cell.
  • Plasmid PHP94810 contains a polynucleotide encoding a first WUSCHEL2 (Wus2) expression cassette operably linked to a chemically inducible regulatory element comprising an Agrobacterium nopaline synthase (NOP) promoter and one operator sequence, a second BABY BOOM (Bbni) expression cassette operably linked to a chemically inducible regulatory element comprising a Zea mays ubiquitin promoter and three operator sequences, a third CYCLIN D2-like (ZM-CYCD2) expression cassette operably linked to a chemically inducible regulatory element cauliflower mosaic virus promoter and three operator sequences, and a fourth ethametsulfuron repressor protein (ESR (LI 5-20)) expression cassette operably linked to and a constitutive promoter.
  • WUSCHEL2 Wus2
  • Bbni BABY BOOM
  • ZM-CYCD2 CYCLIN D2-like expression cassette operably linked to a chemically inducible
  • the three ethametsulfuron inducible expression cassettes are repressed in response to binding of the constitutively expressed ESR (LI 5 -20) repressor protein binding to operator sequences within the regulatory elements.
  • the three ethametsulfuron inducible expression cassettes are de-repressed in response to interactions of the expressed ESR (LI 5-20) repressor protein and ethametsulfuron, a ligand.
  • Plasmid PHP110677 contains a T-DNA having a polynucleotide containing a first expression cassette encoding a CAS polypeptide operably linked to a constitutively expressed Zea mays ubiquitin promoter and a second expression cassette encoding guide RNAs that target a desired target site, ZM-TARGET-GENE-X-CR10 and ZM-TARGET-GENE-X-CR11, used for recruiting double strand break activity, and thus, genome editing, to the target gene locus Gene-X encoding the Gene-XTARGET-GENE-X) polypeptide.
  • this co-transformation method can be performed using expression cassettes containing a plurality of gRNA to produce a plant having a plurality of genome edits or modifications.
  • a second advantage of this co-transformation is the easy exchangeability of one plasmid, for example plasmid PHP 110677 as shown here, with an alternative plasmid encoding different gRNA sequences to produce multiple populations having multiple, different genomic edits.
  • the Fl hybrid embryos (approximately 1.8 mm length) were transformed using an Agrobacterium mixture containing 10% of the Agrobacterium comprising plasmid PHP88158, 45% of the Agrobacterium comprising plasmid PHP94810, and 45% of the Agrobacterium comprising plasmid PHP110677.
  • Stably transformed Fl plants derived from the transformed Fl hybrid embryos, were regenerated, transplanted to soil, and grown under standard conditions. [0380] Testing of the stably transformed Fl plants showed evidence of gene editing.
  • Fl hybrid plant with T-DNA from plasmids PHP94810 and PHP 110677 that lacked evidence for PHP88158 T-DNA integration was sampled and tested for genome modification at the ZM- TARGET-GENE-X-CR10 target site.
  • Evidence for CAS activity was supported by detecting ZM- TARGET-GENE-X-CR10 target mutations, including evidence for a single base insertion at nucleotide 91, a 4 bp deletion at nucleotide 90, and an 8 bp deletion at nucleotide 82.
  • it is expected a proportion of such mutations present in the sporophytic plant cells can become germline heritable in the gametes obtained from the tassel of the plant.
  • the method of the current example can enable editing within a microspore that inherited a wild-type allele at the target site locus given the Cas polypeptide was linked to a constitutively expressed Z. mays ubiquitin promoter.
  • each tassel donor contains a population of gametes that can be gene edited before, during, or after meiosis.
  • each microspore is expected to represent a gametic product of meiosis, therefore, it is anticipated that each regenerated plant comprises a unique individual within this INBRED-A ⁇ INBRED-B breeding population. Since it is expected the current method provides an improved capability to generate a paternally -derived doubled haploid populations using relatively fewer donor plants and with less labor, the current method therefore improves productivity.
  • the method of Example 9 describes a transformation method providing different components using individual plasmids.
  • One benefit is a unique mixture of plasmids can allow providing a combination of unique gRNA to obtain a population inheriting a desired combination of gene edited mutant alleles.
  • One limitation of the above method can be reduced regeneration frequencies due to partial de-repression, often called “leaky” repression, even in the absence of the ligand while under non-induced conditions.
  • Another issue associated with such leaky repression can be negative pleiotropic effects on morphological development of the transplanted plant being grown to maturity.
  • the current example describes an improved plasmid incorporating Cre recombinase activity to enable removal of the plasmid components, preferentially with Cre- mediated excision of T-DNA feature elements that otherwise can negatively impact plant regeneration, plant morphology, or both.
  • the following experiment demonstrates providing simultaneous microspore embryogenesis induction, chromosome doubling, and genome editing activity to a maize haploid cell in vivo to obtain a genome-edited, paternally-derived doubled haploid plant.
  • this paternal trait activity is used in combination with a rapid transformation method (Lowe, K , La Rota, M , Hoerster, G. et al. Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. In Vitro Cell. Dev. Biol. -Plant 54, 240-252 (2018)) and a Cre recombinase for removal of T-DNA components that can negatively impact the plant phenotype.
  • the rapid transformation components of plasmid RV061249 comprises a W s2 expression cassette operably linked by the maize auxin-inducible promoter (Zm- Zxz lpro) in combination with Zm-PLTPpro::Bbm expression cassette allowing healthy, fertile plants to be regenerated.
  • the paternal trait activities used is as described for plasmid PHP94810 in Example 9, including the use of the genetic haploid chromosome doubling factor Zm-CYCD2.
  • genome editing activities are encoded by a CAS polypeptide and ZM-TARGET- GENE-X-CR10 and ZM-TARGET-GENE-X-CR11 guide RNA enabling obtaining plants having gene-X genome modifications.
  • plasmid RV061249 contains a polynucleotide encoding a Cre recombinase expression cassette operably linked to a maize promoter for Heat Shock Protein 17.7.
  • the above feature elements of plasmid RV061249 are flanked by a pair of directional LoxP sites, thereby enabling excision of the aforementioned expression cassettes conferring these combined trait activities.
  • it is expected plants having an excised T- DNA will lack expression of negative pleiotropic effects on morphological development when grown to maturity.
  • PH1V69 is stably transformed plasmid RV061249 and tassels are processed as described in Examples 3 and 4 It is anticipated that CAS/gRNA-mediated editing of the maize genome can provide gene-X edited alleles of microspore-derived plant cells. DNA isolated from the microspore-derived plant cells is used to evaluate the site directed mutation.
  • an Fl breeding cross such as INBRED-A ⁇ INBRED-B
  • INBRED-A ⁇ INBRED-B can be transformed with said tassels to be processed as described in Example 9. It expected the current method will improve the capability to generate a paternally-derived doubled haploid populations using relatively fewer donor plants and with less labor. Second, it is expected the regenerated DO plants will lack any loss of fitness conferred by leaking expression of any plasmid RV061249 expression cassettes following Cre-mediated excision of the T-DNA derived from plasmid RV061249. Thus, the current method improves not only the productivity, but also the phenotypic quality of the resulting doubled haploid lines lacking negative pleiotropic effects.

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Abstract

La présente invention concerne des procédés et des compositions permettant de générer des cellules végétales haploïdes à génome modifié et des cellules végétales haploïdes doublées à génome modifié.
PCT/US2024/046207 2023-09-12 2024-09-11 Procédés et compositions pour générer des haploïdes doublés paternels à génome modifié WO2025059184A1 (fr)

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