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WO2025034520A2 - Recombinaison ciblée - Google Patents

Recombinaison ciblée Download PDF

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Publication number
WO2025034520A2
WO2025034520A2 PCT/US2024/040615 US2024040615W WO2025034520A2 WO 2025034520 A2 WO2025034520 A2 WO 2025034520A2 US 2024040615 W US2024040615 W US 2024040615W WO 2025034520 A2 WO2025034520 A2 WO 2025034520A2
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Prior art keywords
nucleic acid
scion
sequence specific
plant
sequence
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WO2025034520A3 (fr
Inventor
Yi Jia
Michael Lee NUCCIO
Palak KATHIRIA
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Inari Agriculture Technology Inc
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Inari Agriculture Technology Inc
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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)
    • 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 [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to methods of inducing targeted recombination in a scion that is grafted to a rootstock, wherein the rootstock expresses mRNA encoding a sequence specific endonuclease, a sequence specific binding protein fused to a recombinase, and/or a sequence specific recombinase, wherein the mRNA encoding the sequence specific endonuclease, sequence specific binding protein fused to a recombinase, and/or sequence specific recombinase is fused to a meristem transport segment.
  • transgene-free, edited progeny of a wild type scion can be created by grafting a wild type, non-transgenic hypocotyl as a scion onto rootstock that transgenically express both Cas9 and a guide RNA (gRNA) by inducing mobility of the Cas9 and gRNA from the rootstock to the scion by adding tRNA-like sequence (TLS) motifs to the Cas9 and gRNA transcripts, which causes them to move from roots to the shoot, resulting in editing in the non-transgenic grafted shoot to produce an edited chimeric scion (Yang, L. et al.
  • Virus-mediated genome editing has also been demonstrated, in which gRNAs are delivered via viral vectors (such as via the tobacco rattle virus (TRV)) into leaves of plants that transgenically overexpress Cas9, leading to TRV reconstitution and systematic infection throughout the plant, which can then lead to the production of transgenic seeds containing desired Cas9- induced edits (Mahas, A. et al. (2019) ‘Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System’, Methods in Molecular Biology (Clifton, N.J.), 1917, pp.
  • TRV tobacco rattle virus
  • haploid seed induction such as via haploid inducer lines (Segui-Simarro, J.M., Jacquier, N.M. A. and Widiez, T. (2021) ‘Overview of In Vitro and In Vivo Doubled Haploid Technologies’, Methods in Molecular Biology (Clifton, N.J.), 2287, pp. 3-22).
  • haploidization methods for plants using in vitro methods (such as those involving, e.g., in vitro culture of microspores, anthers, ovaries, ovules, and/or flower buds), in vivo methods (such as those involving, e.g., haploid inducer lines, such as CENH3-based haploid inducer lines), and combinations of in vivo and in vitro methods (such as those involving, e.g., wide crosses and/or pollen treatments).
  • in vitro methods such as those involving, e.g., in vitro culture of microspores, anthers, ovaries, ovules, and/or flower buds
  • in vivo methods such as those involving, e.g., haploid inducer lines, such as CENH3-based haploid inducer lines
  • combinations of in vivo and in vitro methods such as those involving, e.g., wide crosses and/or pollen treatments.
  • Plants do not maintain a population of germ cells throughout their lifetime. Vegetative meristems give rise to floral meristems, which will produce the reproductive organs and gametes. Heritable genome edits in plants therefore require that the edits occur either in the gametes themselves or in the cells of the meristem that will give rise to the gametes.
  • One method of accomplishing this is to deliver a transgene to the genome of the entire plant, which produces genome editing reagents in at least the meristem so as to produce the desired edits.
  • the genome editing reagents may include a sequence specific endonuclease, a sequence specific binding protein fused to a recombinase, and/or a sequence specific recombinase.
  • RNAs can be targeted to the shoot apical meristem by the addition of meristem transport segments (Kehr and Buhtz J Exp Bot 2008, 59: 85-92; Ham and Lucas Annu Rev Plant Biol 2017, 68: 173-195; Kehr and Kragler New Phytol 2018, 218: 29-40; Kehr et al. Annu Rev Plant Biol 2022, 73: 457-474). It has been demonstrated that sequences derived from the Arabidopsis FT transcript are capable of targeting a heterologous, non- mobile RNA to the shoot apical meristem (Li et al.
  • RNA encoding genome editing reagents is produced in one part of the plant, loaded into the phloem, and transported to the shoot apical meristem where it is translated and assembled into mature ribonucleoproteins (RNPs) to perform genome editing in meristem nuclei which will eventually form the plant reproductive structures.
  • RNPs ribonucleoproteins
  • Heritable edits are the result.
  • this method is still limited to species that are amenable to transformation.
  • a recent method to introduce germline edits is to target genome editing reagents, including an RNA-guided nuclease and at least one corresponding guide RNA, to the shoot apical meristem (Imai et al. Plant Biotechnol 2020, 37(2): 171-176).
  • This can be achieved through constitutive expression of the nuclear- localized CRISPR Cas nuclease using highly active promoters like those based on ubiquitin genes or CaMV 35S, and expression of the guide RNA(s) from RNA polymerase III promoters (Hassan et al. Trends Plant Sci 2021, 26: 1133-1152).
  • Guide RNAs can be expressed from a constitutive RNA polymerase II promoter if flanked by self-cleaving ribozymes that remove 5’- and 3’-flanking sequence (Tang et al. Plant Biotechnol J 2019, 17: 1431-1445). It is also possible to directly express both the CRISPR Cas nuclease and guide RNAs in the shoot apical meristem using promoters that are highly active in those cells alone (Jackson et al. Development 1994, 120: 405-413). All these approaches require direct expression of the genome editing reagents in the cells to be edited, which limits direct editing to germplasm that can be transformed using routine methods such as Agrobacterium (Altpeter et al.
  • Grafting is a plant procedure in which one plant part from a first genetic donor is functionally fused with a second plant part from a second, and distinct, genetic donor (Bezdicek et al. Agron J 1972, 64: 558-558; Cao et al.
  • Crop Pasture Sci 2019, 70: 585-594 A common use for grafting is to join a rootstock that confers a trait beneficial to growth and/or survival (e.g. robust disease resistance) with a shoot (or scion) that produces high quality fruit. Grafting has been historically quite successful in dicot species and some trees but has only been recently demonstrated in monocots (Reeves et al. Nature 2022, 602: 280- 286). A hallmark of successful grafting is vascular mobility and transmission through a graft junction. Materials loaded into the plant vascular system in the rootstock can be transmitted through the graft junction to the plant scion, and vice versa.
  • Genome editing of commercial crops is limited by the well-known general recalcitrance to transformation of the elite materials. Editing experimental materials and crossing the edits into elite germplasm takes many generations, and the eventual edited phenotype is not predictable. A simple “one step” process for making genome-edited seeds of elite materials would save time and money, enlarging the capacity of a plant editing pipeline to make edits and observe phenotypes in genetic backgrounds of commercial relevance.
  • CRISPR technology for editing the genes of eukaryotes is disclosed in U.S. Patent Application Publications 2016/0138008 Al (now U.S. Pat. No. 10,227,11) and US2015/0344912A1, and in U.S. Pat. Nos.
  • Cpfl (Casl2a) endonucleases and corresponding guide RNAs and PAM sites are disclosed in U.S. Pat. No. 9,790,490 and U.S. patent application Ser. No. 15/566,528 (national phase of PCT Application PCT/EP2016/058442, published as WO 2016/166340), now published as U.S. Patent Application Publication 2018/0282713.
  • CRISPR nucleases useful for editing genomes include C2cl and C2c3 (see Shmakov et al. Mol. Cell 2015, 60: 385-397) and CasX and CasY (see Burstein et al. Nature 2016, doi:10.1038/nature21059).
  • Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in U.S. patent application Ser. No. 15/120,110, published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to International Patent Application PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700).
  • Methods of using CRISPR technology for genome editing in plants are disclosed in in U.S. Patent Application Publications U.S.
  • the present disclosure provides methods of inducing targeted recombination in a scion that is grafted to a rootstock comprising nucleic acid encoding a sequence specific endonuclease, a sequence specific binding protein fused to a recombinase, and/or a sequence specific recombinase, wherein the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein fused to a recombinase, and/or sequence specific recombinase is fused to a meristem transport segment, and plants and seeds produced therefrom.
  • a method of inducing targeted recombination in a scion comprising: a) providing a rootstock expressing a sequence specific endonuclease, wherein a nucleic acid encoding the sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • the mRNA encoding the sequence specific endonuclease is fused to an MTS.
  • the sequence specific endonuclease is a zinc finger nuclease (ZFN), a TALEN or a Cas protein.
  • the sequence specific endonuclease is a Cas protein.
  • a method of inducing targeted recombination in a scion comprising: a) providing a rootstock expressing a sequence specific binding protein, wherein the sequence specific binding protein is fused to a recombinase, and wherein a nucleic acid encoding the sequence specific binding protein fused to the recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific binding protein binds to a target sequence in a genomic locus of the scion; wherein the recombinase induces cleavage and/or recombination in the genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • the mRNA encoding the sequence specific binding protein fused to a recombinase is fused to an MTS.
  • the sequence specific binding protein is selected from the group consisting of: inactive zinc finger nuclease (ZFN) proteins, inactive TALEN proteins, catalytically inactive Cas proteins, and transcription factors.
  • the sequence specific binding protein comprises one or more domains of a catalytically inactive Cas protein.
  • the method further includes delivering a guide nucleic acid that is complementary to the target sequence in the scion.
  • a method of inducing targeted recombination in a scion comprising: a) providing a rootstock expressing a sequence specific recombinase, wherein a nucleic acid encoding the sequence specific recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific recombinase of the rootstock binds to a recombinase site in the genome of the scion; wherein the recombinase induces cleavage and/or recombination in the genome of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • the mRNA encoding the sequence specific recombinase is fused to an MTS.
  • the scion is not of a uniform or inbred line.
  • the sequence specific recombinase comprises one or more domains of a serine recombinase and/or a tyrosine recombinase.
  • the sequence specific recombinase binds specifically to recombination sites within the genome of the scion.
  • the method includes inducing DNA cleavage and/or recombination in a plurality of plants at different genomic loci, wherein the genetic diversity in the plurality of plants is increased following the cleavage and/or recombination compared to the genetic diversity in the plurality of plants prior to the cleavage and/or recombination.
  • the cleavage is a double- stranded break (DSB), a single- stranded break, a transposase-mediated DNA exchange reaction, or a recombinase-mediated DNA exchange reaction.
  • the induced targeted recombination does not comprise recombination with exogenously provided DNA.
  • the method includes delivering a plurality of different guide nucleic acids, wherein the plurality of different guide nucleic acids is complementary to more than one target sequence in one or more genetic loci of the scion.
  • the target sequences are on different loci on the same chromosome, on different loci on homologous chromosomes, and/or on the same locus on homologous chromosomes.
  • the target sequences are on the same locus on homologous chromosomes.
  • the genomic loci are at least 1 centimorgan (cM) apart from each other on the genome of the scion.
  • the target sequence is within an intergenic region.
  • at least one target sequence is within a regulatory element, an intron, or an exon.
  • the regulatory element is in a promotor.
  • the target sequence is within 50 bp of the 5’ or 3’ end of a gene of interest, wherein the target sequence is within 50 bp of a polymorphism of interest, and/or wherein the target sequence is within a genomic region that has sufficient surrounding 5’ and 3’ identity to allow for recombination.
  • the guide nucleic acid is a gRNA.
  • sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is constitutively expressed in the rootstock.
  • RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported from the rootstock to the scion by the plant vascular system.
  • RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported to the scion through the xylem or the phloem.
  • RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported to the meristem and/or a somatic region of the scion. In some embodiments, RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is translated in the scion. In some embodiments, RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is translated in a meristem cell of the scion and/or a somatic cell of the scion.
  • targeted recombination is induced in a meristem cell and/or a scion cell.
  • the scion and the rootstock are different plant species.
  • the scion and the rootstock are the same plant species.
  • the scion and/or rootstock is a dicot.
  • the scion and/or rootstock is a monocot.
  • the guide nucleic acid is provided by providing a vector that encodes the guide nucleic acid, optionally wherein the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase and the nucleic acid encoding the guide nucleic acid are provided in different vectors.
  • the plurality of different guide nucleic acids are provided in a single vector that encodes each of the guide nucleic acids.
  • the plurality of different guide nucleic acids are provided in multiple different vectors, wherein each vector encodes one or more different guide nucleic acids.
  • the guide nucleic acid or plurality of different guide nucleic acids is delivered to the scion in a viral vector or a T-DNA vector comprising nucleic acid encoding the guide nucleic acid.
  • delivery of the guide nucleic acid or plurality of different guide nucleic acids comprises spraying a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem. In some embodiments, delivery of the guide nucleic acid or plurality of different guide nucleic acids comprises incubating the scion with a composition comprising the guide nucleic acid. In some embodiments, delivery of the guide nucleic acid comprises transformation of the scion by Agrobacterium rhizogenes or Agrobacterium tumefaciens.
  • the MTS is a Flowering Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.
  • the FT-derived sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.
  • the guide nucleic acid or plurality of different guide nucleic acids are fused to a meristem transport segment (MTS).
  • MTS meristem transport segment
  • the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase.
  • the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase.
  • the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is operably linked to a promoter.
  • the promoter is active in roots and/or phloem companion cells.
  • the promoter is the promoter of a gene selected from the group consisting of Arabidopsis WRKY6, chickpea WRKY31, carrot MYB113, com GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof.
  • the promoter is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FORI gene, a rice tungro bacilliform vims promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle virus promoter, a wheat dwarf virus promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloem- specific isoform of plasmamembrane H+-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter.
  • the promoter is a constitutive promoter.
  • the constitutive promoter is a ubiquitin promoter.
  • sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in dicots. In some embodiments, the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in monocots. In some embodiments, the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in corn, soy, or wheat.
  • nucleic acid(s) encoding the guide nucleic acid(s) is operably linked to a promoter.
  • the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.
  • the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the rootstock.
  • the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the scion.
  • nucleic acid(s) encoding the guide nucleic acid(s) and the MTS is/are located between two ribozyme sequences.
  • each of the ribozyme sequences is independently selected from the group consisting of a hammerhead ribozyme sequence, a HDV ribozyme sequence, a Csy4 sequence, and a tRNA processing enzyme sequence.
  • the method comprises delivering two or more, three or more, four or more, or five or more different guide nucleic acids.
  • the two or more, three or more, four or more, or five or more different guide nucleic acids are each joined to an MTS.
  • the Cas is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j.
  • the Cas is a Cas nickase.
  • the Cas nickase is a Cas9 nickase or a Cas 12 nickase.
  • the Cas nickase comprises mutation in one or more nuclease active sites.
  • the rootstock and/or scion further comprises a nucleic acid encoding a detectable marker fused to a nucleic acid encoding a MTS.
  • the guide nucleic acid comprises a 5-methycytosine group.
  • nucleic acid encoding the guide nucleic acid and the MTS further comprises a hammerhead ribozyme sequence 5’ to the nucleic acid encoding the guide nucleic acid and the MTS, and a HDV ribozyme 3’ to the nucleic acid encoding the guide nucleic acid and the MTS.
  • the nucleic acid encoding the guide nucleic acid and the MTS further comprises a terminator.
  • the terminator is a U6 terminator.
  • the method further includes selecting a scion comprising a somatic cell and/or a meristem cell comprising the recombined genomic locus.
  • the method further includes producing a seed, protoplast, or cell culture from the somatic cell and/or the meristem cell of the selected scion, wherein the seed, protoplast, or cell culture comprises the recombined genomic locus.
  • the method further includes regenerating a plant from the seed, protoplast, or cell culture.
  • the method further includes retrieving a progeny of the scion, wherein the genome of the progeny comprises the recombined genomic locus.
  • the guide nucleic acid further comprises: (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide nucleic acid; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide nucleic acid; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar.
  • each of the one or more modified nucleotides is independently selected from the group consisting of a 2'-O-methyl nucleotide, a 2'-O-methyl-3'-phosphorothioate nucleotide, a 2'-O-methyl-3'-phosphonoacetate nucleotide, and a 2'-O-methyl-3'- phosphonothioacetate nucleotide.
  • the one or more modified nucleotide comprises a modified internucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group.
  • the composition comprising the guide nucleic acid comprises a surfactant.
  • the composition comprising the guide nucleic acid comprises glass beads coated with the guide nucleic acid.
  • delivery of the guide nucleic acid comprises rubbing a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • delivery of the guide nucleic acid comprises injecting a composition comprising the guide nucleic acid into the stem of the scion.
  • delivery of the guide nucleic acid comprises leaf infiltration of a composition comprising the guide nucleic acid into the leaf.
  • the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • the composition comprising the guide nucleic acid comprises a nuclease inhibitor.
  • the nuclease inhibitor comprises an RNase inhibitor.
  • delivery of the guide nucleic acid comprises biolistic transformation of nucleic acid encoding the guide nucleic acid into the leaf, shoot, stem, and/or meristem.
  • the biolistic transformation comprises transformation of circular DNA encoding the guide nucleic acid.
  • the plurality of different guide nucleic acids comprises two or more guide RNAs that are encoded by a single precursor RNA.
  • the two or more guide RNAs are each flanked by a direct repeat.
  • the method further includes: c) inducing the formation of haploid seeds; d) producing double haploid lines from said haploid seeds; and optionally e) genotyping and/or characterizing the double haploid lines for recombination profiling.
  • the method further includes: c) producing one or more F2 lines from the scion by allowing the scion to self-pollinate; d) selecting an F2 line comprising a desired recombination; and optionally e) producing additional lines from the selected F2 line by haploid production and/or genome doubling.
  • the scion is from an Fl plant. In some embodiments, the recombination stacks beneficial alleles from the parents of Fl plant into a single progeny. [0042] In some embodiments, the scion is diploid. In some embodiments, the scion is haploid. In some embodiments, the scion and/or rootstock is from a soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana or wheat plant. In some embodiments, the scion and/or rootstock is from a soy, maize, or wheat plant.
  • the method further includes inducing targeted recombination in a scion of the progeny by subjecting the scion of the progeny to the methods of any of the preceding embodiments to produce one or more additional recombined loci. In some embodiments, the method further includes retrieving one or more progeny comprising the one or more additional recombined loci. In some embodiments, the method further includes inducing targeted recombination in a scion of the one or more progeny by subjecting the scion of the one or more progeny to the methods of any of the preceding embodiments to produce one or more other recombined loci.
  • provided herein is a plant produced by the method of any of the preceding embodiments.
  • a seed produced by the plant produced by the method of any of the preceding embodiments is plant grown from a seed produced by the plant produced by the method of any of the preceding embodiments.
  • provided herein is a plant genome produced by the targeted recombination of the method of any of the preceding embodiments.
  • a plant genome produced by the targeted recombination of the method of any of the preceding embodiments wherein the plant genome is recombined at the target sequence compared to a corresponding genome that was not subjected to the method.
  • provided herein is an isolated plant cell produced by the method of any of the preceding embodiments.
  • a plant comprising a scion grafted onto a rootstock, wherein the rootstock comprises: a heterologous sequence specific endonuclease and/or a nucleic acid encoding a heterologous sequence specific nuclease, wherein a nucleic acid encoding the sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS); a heterologous sequence specific binding protein and/or a nucleic acid encoding a heterologous sequence specific binding protein, wherein the sequence specific binding protein is fused to a recombinase, and wherein a nucleic acid encoding the sequence specific binding protein fused to the recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and/or a heterologous sequence specific recombinase and/or a nucleic acid encoding a heterologous sequence specific nucle
  • allelic variant refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.
  • the term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as "A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the phrase “codon optimization” refers to the process of modifying a nucleic acid sequence for use in a desired host kingdom, phylum, class, order, family, genus, or species, by replacing at least one codon of the nucleic acid with codons that are more frequently used in the genes of the desired host kingdom, phylum, class, order, family, genus, or species, without alteration of the amino acid sequence encoded by the nucleic acid.
  • the term “complementary” refers to sequences with at least sufficient complementarity to permit enough base-paring for two nucleic acids to hybridize (for example, for a tether to hybridize with or bind to a gRNA or donor DNA), which in some examples may be under typical physiological conditions for the cell.
  • the oligonucleotide or polynucleotide is at least 80% complementary to the target, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target.
  • complex refers to two or more associated components, such as two or more associated nucleic acids and/or proteins.
  • a complex may include two or more covalently linked nucleic acids and/or proteins, two or more non-covalently linked nucleic acids and/or proteins, or a combination thereof.
  • an "elite plant line” is any plant variety produced by breeding and/or modem biotechnological tools and selected for superior agronomic performance.
  • the terms “comprise,” comprises, “comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
  • CRISPR-Cas nuclease and “Cas nuclease” are used interchangeably herein to refer to the same grouping of RNA directed nucleases.
  • engineered means artificial, synthetic, or not occurring in nature.
  • a polynucleotide that includes two DNA sequences that are heterologous to each other can be engineered or synthesized by recombinant nucleic acid techniques.
  • genomic locus refers to a location on a chromosome, such as, for instance, one or more nucleotides, a gene or portion thereof, a gene cluster, or any other definable region within a genome.
  • a genomic locus may be defined by, for instance, a specific sequence, flanking markers, or linkage distance in relation to other sequences or genes.
  • a graft As used herein, the terms “a graft,” “to graft,” and “grafting” refer to the technique wherein two plants are joined by their vasculature such that they fuse to form a single grafted plant.
  • the plant that maintains or will maintain the root system after grafting is referred to herein as the “rootstock”.
  • the plant grafted onto the rootstock is referred to herein as the “shoot”, “plant scion” or “scion”.
  • Grafting includes “micrografting” (Pena et al. Plant Cell Rep 1995, 14: 616-619; CN105519434A; CN110178564A), “minigrafting” (Marques et al. Sci Hortic 2011, 129: 176-182), and other forms of grafting known to those in the art.
  • heterograft refers to a graft between a rootstock and a scion of different species.
  • homograft refers to a graft between a rootstock and a scion of the same species.
  • the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
  • the phrase “meristem transport segment” or “MTS” refers to an RNA tag that, when fused to another RNA molecule, results in delivery of the RNA fusion molecule to the meristem of the plant.
  • the term “mobile” refers to the ability of a molecule or a collection of molecules to move within the plant.
  • a fusion of a nucleic acid encoding a Cas nuclease and a meristem transport segment (MTS) results in a mobile Cas, which is capable of being transported through the plant vascular system to the meristem of the plant, including through a graft junction.
  • a fusion of an RNA molecule and a meristem transport segment (MTS) results in a “mobile RNA”, which is capable of being transported through the plant vascular system to the meristem of the plant, including through a graft junction.
  • RNA molecule comprising a “meristem transport sequence” (MTS) is operably linked or fused to a guide RNA if the MTS provide for delivery of the guide RNA to meristem cells.
  • MTS meristem transport sequence
  • orthologous or “orthologue” are used to describe genes or the RNAs or proteins encoded by those genes that are from different species but which have the same function (e.g., encode RNAs which exhibit the same meristem transport function). Orthologous genes will typically encode RNAs or proteins with some degree of sequence identity and can also exhibit conservation of sequence motifs, and/or conservation of structural features including RNA stem loop structures.
  • the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant.
  • plant parts include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
  • some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
  • substantially purified defines an isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition.
  • substantially purified RNA molecule is used herein to describe an RNA molecule which has been separated from other contaminant compounds including, but not limited to polypeptides, lipids, and carbohydrates.
  • a substantially purified RNA is at least 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% free of contaminating compounds by weight.
  • RNA molecule can be combined with other compounds including buffers, RNase inhibitors, surfactants, and the like in a composition.
  • polynucleotide refers to a nucleic acid molecule containing multiple nucleotides and encompasses both “oligonucleotides” (defined here as a polynucleotide molecule of between 2-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Polynucleotides are generally described as single- or double-stranded.
  • polynucleotide contains double-stranded regions formed by intra- or intermolecular hybridization
  • the length of each double- stranded region is conveniently described in terms of the number of base pairs.
  • aspects of this invention include the use of polynucleotides or compositions containing polynucleotides; embodiments include one or more oligonucleotides or polynucleotides or a mixture of both, including single- or double-stranded RNA or single- or double-stranded DNA or double- stranded DNA/RNA hybrids or chemically modified analogues or a mixture thereof.
  • a polynucleotide includes a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine.
  • the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein Annu. Rev. Biochem.
  • oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate intemucleotide linkage modifications; modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis; and oligonucleotides or polynucleotides can be labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope).
  • fluorescent moiety e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels
  • other label e.g., biotin or an isotope.
  • chromosomal crossovers refers to a process by which two DNA molecules exchange nucleotide sequences.
  • the two DNA molecules that undergo recombination are two sets of parental chromosomes, such as, for instance, a parental chromosome contributed by a male parent and a parental chromosome contributed by a female parent.
  • the two DNA molecules that undergo recombination are homologous chromosomes.
  • the two DNA molecules that undergo recombination are homologous DNA sequences on two different chromosomes or homoeologous sequences on two different chromosomes (i.e., a pair of sequences that originated by speciation and regrouped into the same genome by allopolyploidization).
  • the two DNA molecules that undergo recombination are homologous chromosomes.
  • the two DNA molecules that undergo recombination are non-homologous chromosomes.
  • the two DNA molecules that undergo recombination are homoeologous chromosomes, i.e., chromosomes of different species that share an ancestral origin, such as, for instance, in a scion derived from an allopolyploid plant (for a description of allopolyploidy in various plant crops, see, e.g., Osabe, K., et al. (2012) Multiple mechanisms and challenges for the application of allopolyploidy in plants. Int J Mol Sci. 2012;13(7):8696-8721. doi: 10.3390/ijms 13078696. Epub 2012 Jul 13. PMID: 22942729; PMCID: PMC3430260).
  • recombination event refers to a single instance of recombination occurring between two DNA molecules. Recombination events may comprise, for example, homologous recombination, non-homologous recombination, sisterchromatid exchange, multiple chromosome rearrangements, and symmetric and asymmetric recombination, any of which may take place in a genic sequence or in an intergenic sequence.
  • sequence identity refers to the percent similarity of two polynucleotides or polypeptides.
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available at ncbi[dot]nlm[dot]nih[dot]gov/BLAST. See, e.g., Altschul et al. Mol. Biol. 1990, 215:403-410.
  • FASTA Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol., 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See Mol. Biol., 48: 443-453 (1970).
  • target sequence refers to a genomic sequence that is bound by a sequence specific endonuclease, sequence specific binding protein fused to a recombinase as described herein.
  • a recombination event at a target sequence is referred to herein as a “targeted recombination event” or a “directed chromosomal crossover”.
  • vascular system or “vasculature” refer to the transport systems within the plant. This includes xylem, phloem, and cambium.
  • T-DNA or “transfer DNA” refer to the DNA transferred from the tumor-inducing plasmid of species of bacteria such as, but not limited to, Agrobacterium tumefaciens and Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), to the nuclear genome of a host plant.
  • Agrobacterium tumefaciens and Agrobacterium rhizogenes (also known as Rhizobium rhizogenes)
  • Rhizobium rhizogenes also known as Rhizobium rhizogenes
  • T-DNA vector refers to a transfer DNA vector system comprising as least a disarmed tumor inducing (Ti) plasmid of species of bacteria such as, but not limited to, Agrobacterium tumefaciens and Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), containing a T-DNA and a vector backbone, and a helper plasmid containing vir virulence genes.
  • a T-DNA vector system may be a binary vector system; a superbinary vector system wherein the Ti plasmid also comprises virulence genes (Komari et al.
  • nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art.
  • A. Editing of a grafted scion mediated by root expression of a sequence specific endonuclease provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a sequence specific endonuclease that is fused to a nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • the sequence specific endonuclease is a zinc finger nuclease (ZFN).
  • the sequence specific endonuclease is a TALEN protein. In some embodiments, the sequence specific endonuclease is a Cas protein. In some embodiments, the method includes delivering a guide nucleic acid that is complementary to a target sequence in the scion.
  • the rootstock expresses a nucleic acid encoding a Cas nuclease and the scion expresses a guide nucleic acid. In some embodiments, the rootstock expresses a nucleic acid encoding a Cas nuclease and a guide nucleic acid. In some embodiments, the rootstock expresses a nucleic acid encoding a Cas nuclease, and a guide nucleic acid is delivered to the scion. In some embodiments, the rootstock expresses a nucleic acid encoding a Cas nuclease, and a guide nucleic acid is delivered to the rootstock.
  • the guide nucleic acid encodes a guide RNA for the Cas nuclease. In some embodiments, the guide nucleic acid and/or the nucleic acid encoding the Cas nuclease is fused to a nucleic acid encoding a MTS.
  • the rootstock expresses a nucleic acid encoding a ZFN. In some embodiments, the rootstock expresses a nucleic acid encoding a TALEN.
  • a rootstock provides nucleic acid encoding sequence specific endonucleases, such as, e.g., a zinc finger nuclease (ZFN), a TALEN, or a Cas nuclease, to the plant vascular system.
  • ZFN zinc finger nuclease
  • TALEN a zinc finger nuclease
  • Cas nuclease Cas nuclease
  • RNA encoding a sequence specific endonuclease is transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding a sequence specific endonuclease is translated in the scion. In some embodiments, a meristem of the scion is edited. [0082] Provided herein is a rootstock comprising nucleic acid encoding a sequence specific endonuclease, wherein the nucleic acid is fused to nucleic acid encoding a meristem transport segment (MTS).
  • MTS meristem transport segment
  • a scion comprising a guide nucleic acid that guides a sequence specific endonuclease such as a Cas nuclease that is fused to the MTS and expressed by the rootstock to a target sequence in the genome of the scion.
  • a sequence specific endonuclease such as a Cas nuclease that is fused to the MTS and expressed by the rootstock to a target sequence in the genome of the scion.
  • the nucleic acid encoding the sequence specific endonucleases are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots, o Agrobacterium tumefaciens.
  • Agrobacterium rhizogenes also known as Rhizobium rhizogenes
  • the nucleic acid encoding sequence specific endonucleases and a guide nucleic acid are provided in the same vector.
  • the nucleic acid encoding the sequence specific endonucleases and a guide nucleic acid are provided in different vectors.
  • the nucleic acid encoding the guide nucleic acid is provided to the scion by Agrobacterium tumefaciens.
  • the vector is a viral vector.
  • the vector is a T-DNA vector.
  • the vector is a viral vector or a T-DNA vector.
  • the rootstock and/or scion comprises nucleic acid encoding two or more, three or more, four or more, or five or more guide nucleic acids.
  • the nucleic acid encoding each of the two or more, three or more, four or more, or five or more guide nucleic acids is joined to an MTS.
  • the guide nucleic acid is a guide RNA.
  • the guide nucleic acid is a guide DNA.
  • the guide nucleic acid comprises both DNA and RNA.
  • the rootstock further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.
  • a scion is grafted onto the rootstock.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific endonuclease results in the sequence specific endonuclease being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction.
  • Nucleic acid encoding the sequence specific endonuclease are translated in the cytosol of cells of the scion meristem and imported into meristem nuclei, whereupon the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.
  • editing of the scion meristem can be accomplished without the introduction of a transgene to the genome of the scion.
  • the scion and resulting progeny will undergo targeted recombination without containing sequences encoding the sequence specific endonucleases expressed from the rootstock in its genome.
  • This will result in targeted recombination that has the effect of decoupling the genetic gain available in the genome of the scion from the limited frequency of naturally occurring random recombination events by not only increasing the number of recombination events but also targeting recombination events to specific, desired loci in the genome of the scion.
  • the provided methods will also result in faster breeding and safety programs, as there is no possibility of off-target effects from insertion of a transgene into an inopportune location in the genome, and there is no need for additional breeding or selection to remove a transgene encoding sequence specific endonucleases from the scion genome.
  • the provided line of rootstocks comprising nucleic acid encoding a sequence specific endonuclease can be a modular tool for inducing recombination in a number of existing elite plant lines. A single rootstock line can be used to induce recombination in many grafted scions, without the need to transform each scion.
  • the provided methods will enlarge the capacity of a plant editing pipeline to increase genetic gain in genetic backgrounds of commercial relevance.
  • the present application provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a sequence specific binding protein fused to a recombinase that is fused to a nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein binds to a target sequence in a genomic locus of the scion, and wherein the recombinase induces cleavage and/or recombination in the genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • the sequence specific binding protein is selected from the group consisting of: inactive zinc finger nuclease (ZFN) proteins, inactive TALEN proteins, catalytically inactive Cas proteins, and transcription factors.
  • the rootstock expresses a nucleic acid encoding a sequence specific binding protein fused to a recombinase and the scion expresses a guide nucleic acid.
  • the rootstock expresses a nucleic acid encoding a sequence specific binding protein fused to a recombinase and a guide nucleic acid.
  • the rootstock expresses a nucleic acid encoding a sequence specific binding protein fused to a recombinase, and a guide nucleic acid is delivered to the scion.
  • the rootstock expresses a nucleic acid encoding a sequence specific binding protein fused to a recombinase, and a guide nucleic acid is delivered to the rootstock.
  • the guide nucleic acid encodes a guide RNA for a sequence specific binding protein fused to a recombinase, wherein the sequence specific binding protein comprises one or more domains of a catalytically inactive Cas protein.
  • the guide nucleic acid and/or the nucleic acid encoding the sequence specific binding protein fused to a recombinase is fused to a nucleic acid encoding a MTS.
  • the rootstock expresses a nucleic acid encoding a sequence specific binding protein fused to a recombinase.
  • a rootstock provides nucleic acid encoding sequence specific binding protein fused to a recombinase to the plant vascular system.
  • RNA encoding a sequence specific binding protein fused to a recombinase are transported from the rootstock to the scion by the plant vascular system.
  • RNA encoding a sequence specific binding protein fused to a recombinase are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding a sequence specific binding protein fused to a recombinase is translated in the scion. In some embodiments, a meristem of the scion is edited.
  • a rootstock comprising nucleic acid encoding a sequence specific binding protein fused to a recombinase, wherein the nucleic acid is fused to nucleic acid encoding a meristem transport segment (MTS).
  • a scion comprising a guide nucleic acid that guides the sequence specific binding protein fused to a recombinase that is fused to the MTS and expressed by the rootstock to a target sequence in the genome of the scion.
  • the nucleic acid encoding the sequence specific binding protein fused to a recombinase are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots, o Agrobacterium tumefacien.
  • Agrobacterium rhizogenes also known as Rhizobium rhizogenes
  • the nucleic acid encoding the sequence specific binding protein fused to a recombinase and a guide nucleic acid are provided in the same vector.
  • the nucleic acid encoding the sequence specific binding protein fused to a recombinase and a guide nucleic acid are provided in different vectors.
  • the nucleic acid encoding the guide nucleic acid is provided to the scion by Agrobacterium tumefaciens.
  • the vector is a viral vector.
  • the vector is a T-DNA vector.
  • the vector is a viral vector or a T-DNA vector.
  • the rootstock and/or scion comprises nucleic acid encoding two or more, three or more, four or more, or five or more guide nucleic acids.
  • the nucleic acid encoding each of the two or more, three or more, four or more, or five or more guide nucleic acids is joined to an MTS.
  • the guide nucleic acid is a guide RNA.
  • the guide nucleic acid is a guide DNA.
  • the guide nucleic acid comprises both DNA and RNA.
  • the rootstock further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.
  • a scion is grafted onto the rootstock.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific binding protein fused to a recombinase results in the sequence specific binding protein fused to a recombinase being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction.
  • Nucleic acid encoding the sequence specific binding protein fused to a recombinase are translated in the cytosol of cells of the scion meristem and imported into meristem nuclei, whereupon the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.
  • editing of the scion meristem can be accomplished without the introduction of a transgene to the genome of the scion.
  • the scion and resulting progeny will undergo targeted recombination without containing sequences encoding the sequence specific binding protein fused to a recombinase in its genome.
  • This will result in targeted recombination that has the effect of decoupling the genetic gain available in the genome of the scion from the limited frequency of naturally occurring random recombination events by not only increasing the number of recombination events but also targeting recombination events to specific, desired loci in the genome of the scion.
  • the provided methods will also result in faster breeding and safety programs, as there is no possibility of off-target effects from insertion of a transgene into an inopportune location in the genome, and there is no need for additional breeding or selection to remove a transgene encoding the sequence specific binding protein fused to a recombinase from the scion genome.
  • the provided line of rootstocks comprising the sequence specific binding protein fused to a recombinase can be a modular tool for inducing recombination in a number of existing elite plant lines. A single rootstock line can be used to induce recombination in many grafted scions, without the need to transform each scion.
  • the provided methods will enlarge the capacity of a plant editing pipeline to increase genetic gain in genetic backgrounds of commercial relevance.
  • the present application provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a sequence specific recombinase that is fused to a nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase of the rootstock binds to a recombinase site in the genome of the scion, and wherein the recombinase induces cleavage and/or recombination in the genome of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • a rootstock provides nucleic acid encoding a sequence specific recombinase to the plant vascular system.
  • RNA encoding a sequence specific recombinase are transported from the rootstock to the scion by the plant vascular system.
  • RNA encoding a sequence specific recombinase are transported from the rootstock to the scion through the phloem.
  • RNA encoding a sequence specific recombinase is translated in the scion.
  • a meristem of the scion is edited.
  • a rootstock comprising nucleic acid encoding a sequence specific recombinase, wherein the nucleic acid is fused to nucleic acid encoding a meristem transport segment (MTS).
  • MTS meristem transport segment
  • the nucleic acid encoding the sequence specific recombinase are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots, o Agrobacterium tumefaciens.
  • the nucleic acid encoding the sequence specific recombinase are provided in a vector.
  • the vector is a viral vector.
  • the vector is a T-DNA vector.
  • the vector is a viral vector or a T-DNA vector.
  • the rootstock further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.
  • a scion is grafted onto the rootstock.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific recombinase results in the sequence specific recombinase being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction.
  • Nucleic acid encoding the sequence specific recombinase are translated in the cytosol of cells of the scion meristem and imported into meristem nuclei, whereupon the genome of the meristem nuclei is edited.
  • Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. [0100]
  • editing of the scion meristem can be accomplished without the introduction of a transgene to the genome of the scion.
  • the scion and resulting progeny will undergo targeted recombination without containing sequences encoding the sequence specific recombinase in its genome.
  • the provided line of rootstocks comprising the nucleic acid encoding the sequence specific recombinase can be a modular tool for inducing recombination in a number of existing elite plant lines.
  • a single rootstock line can be used to induce recombination in many grafted scions, without the need to transform each scion.
  • the provided methods will enlarge the capacity of a plant editing pipeline to increase genetic gain in genetic backgrounds of commercial relevance.
  • a guide nucleic acid is not used to target a sequence specific endonuclease to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • the sequence specific endonuclease does not require a guide nucleic acid for targeting to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • a guide nucleic acid targets a sequence specific endonuclease to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • the present application thus also provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a sequence specific endonuclease, wherein the nucleic acid encoding the sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the scion a guide RNA for the sequence specific endonuclease.
  • MTS meristem transport segment
  • the sequence specific endonuclease is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhiz.ogenes). producing a plant with transgenic hairy roots, or Agrobacterium tumefaciens.
  • a rootstock provides nucleic acid encoding a sequence specific endonuclease to the plant vascular system.
  • a scion is grafted onto the rootstock.
  • the method comprises delivering two or more, three or more, four or more, or five or more guide nucleic acids.
  • the guide nucleic acids are guide RNAs.
  • the guide nucleic acids are guide DNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide nucleic acids are each joined to an MTS. In some embodiments, two or more guide nucleic acids are encoded by a single precursor nucleic acid, such as a precursor RNA. In some embodiments, the two or more guide nucleic acids are each flanked by a direct repeat.
  • a guide nucleic acid may be delivered to the meristem in a variety of ways.
  • the guide nucleic acid is delivered to the scion or directly to the meristem of the scion.
  • the guide nucleic acid is delivered to the rootstock and transported into the scion.
  • the guide nucleic acid is produced in vitro.
  • the guide nucleic acid is methylated in vitro, such as by an RNA or DNA methylase, to promote mobility.
  • the guide nucleic acid is fused to a meristem transport segment (MTS).
  • MTS meristem transport segment
  • Delivery of the guide nucleic acid can occur through the following non-exhaustive list: through use of an RNA or DNA spray comprising the guide nucleic acid and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by application of a composition comprising the guide nucleic acid onto a leaf after rubbing the leaf with 200 grit sandpaper with a dowel; by spraying onto a leaf very fine glass beads coated with a composition comprising the guide nucleic acid; by injection of a composition comprising the guide nucleic acid into the stem; by infiltration of the leaf with a composition comprising the guide nucleic acid; by direct uptake in the roots of a composition comprising the guide nucleic acid; or by biolistic delivery to leaves or other tissue with circular DNA expressing the guide nucleic acid.
  • delivery of the guide nucleic acid comprises spraying a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • the composition comprising the guide nucleic acid comprises a surfactant.
  • the composition comprising the guide nucleic acid comprises glass beads coated with the guide nucleic acid.
  • delivery of the guide nucleic acid comprises rubbing a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • delivery of the guide nucleic acid comprises injecting a composition comprising the guide nucleic acid into the stem.
  • delivery of the guide nucleic acid comprises leaf infiltration of a composition comprising the guide nucleic acid into the leaf.
  • the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • the composition comprising the guide nucleic acid comprises a nuclease inhibitor.
  • the nuclease inhibitor comprises an RNase inhibitor and/or a DNase inhibitor.
  • delivery of the guide nucleic acid comprises biolistic transformation of the guide nucleic acid or of nucleic acid encoding the guide nucleic acid, such as nucleic acid encoding guide RNA, into the leaf, shoot, stem, and/or meristem.
  • the biolistic transformation comprises transformation of circular DNA encoding guide RNA.
  • RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported by plant vascular system. In some embodiments, RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported to the scion through the xylem or the phloem. In some embodiments, RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported to the meristem. In some embodiments, RNA encoding the sequence specific endonuclease is translated in the meristem.
  • the meristem is edited.
  • the guide nucleic acid is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly.
  • the guide nucleic acid is imported into the meristem nuclei.
  • the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.
  • the provided methods allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the genome of the edited plant scion. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on rootstock plants providing the sequence specific endonuclease, and different guide nucleic acids can be delivered to the different plant scions.
  • this allows for direct 1 comparison of the results of providing different guide RNAs, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide RNAs, and phenotypic changes as a result of edits induced by different guide RNAs.
  • the provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.
  • the provided methods allow for a reduced number of required transformation events.
  • the rootstock providing the sequence specific endonuclease can be used with a wide variety of delivered guide nucleic acids, increasing the modularity of the editing system.
  • a guide nucleic acid is not used to target a sequence specific binding protein fused to a recombinase to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • the sequence specific binding protein fused to a recombinase does not require a guide nucleic acid for targeting to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • a guide nucleic acid targets a sequence specific binding protein fused to a recombinase to a desired locus in the genome of the scion that has been selected for targeted recombination.
  • the present application thus also provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a sequence specific binding protein fused to a recombinase, wherein the nucleic acid encoding sequence specific binding protein fused to a recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the scion a guide RNA for the sequence specific binding protein fused to a recombinase.
  • MTS meristem transport segment
  • the sequence specific binding protein fused to a recombinase is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhiz.ogenes). producing a plant with transgenic hairy roots, or Agrobacterium tumefaciens.
  • a rootstock provides nucleic acid encoding a sequence specific binding protein fused to a recombinase to the plant vascular system.
  • a scion is grafted onto the rootstock.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific binding protein fused to a recombinase results in the nucleic acid encoding the sequence specific binding protein fused to a recombinase being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction.
  • Nucleic acid encoding the sequence specific binding protein fused to a recombinase is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei.
  • the method comprises delivering two or more, three or more, four or more, or five or more guide nucleic acids.
  • the guide nucleic acids are guide RNAs. In some embodiments, the guide nucleic acids are guide DNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide nucleic acids are each joined to an MTS. In some embodiments, two or more guide nucleic acids are encoded by a single precursor nucleic acid, such as a precursor RNA. In some embodiments, the two or more guide nucleic acids are each flanked by a direct repeat. [0111] A guide nucleic acid may be delivered to the meristem in a variety of ways. For example, in some embodiments, the guide nucleic acid is delivered to the scion or directly to the meristem of the scion.
  • the guide nucleic acid is delivered to the rootstock and transported into the scion. In some embodiments, the guide nucleic acid is produced in vitro. In some embodiments, the guide nucleic acid is methylated in vitro, such as by an RNA or DNA methylase, to promote mobility. In some embodiments, the guide nucleic acid is fused to a meristem transport segment (MTS). Delivery of the guide nucleic acid can occur through the following non-exhaustive list: through use of an RNA or DNA spray comprising the guide nucleic acid and a simple surfactant (see, e.g., U.S. Pat. No.
  • delivery of the guide nucleic acid comprises spraying a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • the composition comprising the guide nucleic acid comprises a surfactant. In some embodiments, the composition comprising the guide nucleic acid comprises glass beads coated with the guide nucleic acid. In some embodiments, delivery of the guide nucleic acid comprises rubbing a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem. In some embodiments, delivery of the guide nucleic acid comprises injecting a composition comprising the guide nucleic acid into the stem. In some embodiments, delivery of the guide nucleic acid comprises leaf infiltration of a composition comprising the guide nucleic acid into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • the composition comprising the guide nucleic acid comprises a nuclease inhibitor.
  • the nuclease inhibitor comprises an RNase inhibitor and/or a DNase inhibitor.
  • delivery of the guide nucleic acid comprises biolistic transformation of the guide nucleic acid or of nucleic acid encoding the guide nucleic acid, such as nucleic acid encoding guide RNA, into the leaf, shoot, stem, and/or meristem.
  • the biolistic transformation comprises transformation of circular DNA encoding guide RNA.
  • RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported by plant vascular system. In some embodiments, RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported to the scion through the xylem or the phloem. In some embodiments, RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported to the meristem. In some embodiments, RNA encoding the sequence specific binding protein fused to a recombinase is translated in the meristem.
  • the meristem is edited.
  • the guide nucleic acid is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly.
  • the guide nucleic acid is imported into the meristem nuclei.
  • the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.
  • the provided methods allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the genome of the edited plant scion. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on rootstock plants providing the sequence specific binding protein fused to a recombinase, and different guide nucleic acids can be delivered to the different plant scions.
  • the provided methods allow for a reduced number of required transformation events.
  • the rootstock providing the sequence specific binding protein fused to a recombinase can be used with a wide variety of delivered guide nucleic acids, increasing the modularity of the editing system.
  • the present application provides methods of editing a genomic target in a plant meristem comprising providing a plant comprising nucleic acid encoding a sequence specific endonuclease, wherein the nucleic acid encoding a sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the root of the plant a guide nucleic acid for the sequence specific endonuclease.
  • the plant comprising the nucleic acid encoding a sequence specific endonuclease is a rootstock.
  • a scion is grafted onto the rootstock.
  • the genomic editing reagents are provided to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenesf producing a plant with transgenic hairy roots.
  • Agrobacterium rhizogenes also known as Rhizobium rhizogenesf producing a plant with transgenic hairy roots.
  • the sequence specific endonuclease is delivered to the plant root by infection with Agrobacterium rhizogenes (also known as Rhizobium rhi ogenes). producing a plant with transgenic hairy roots.
  • the plant provides nucleic acid encoding sequence specific endonuclease to the plant vascular system.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific endonuclease results in the nucleic acid encoding the sequence specific endonuclease being transported to cells of the meristem of the scion through the plant vascular system.
  • the nucleic acid encoding the sequence specific endonuclease is transported from the rootstock to the scion through the graft junction.
  • RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported by plant vascular system.
  • RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported through the xylem or the phloem. In some embodiments, RNA encoding the sequence specific endonuclease and/or the guide nucleic acid is transported to the meristem, wherein the sequence specific endonuclease and/or the guide nucleic acid is translated in the meristem. Nucleic acid encoding the sequence specific endonuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei. [0118] In some embodiments, the guide nucleic acid is delivered to the roots.
  • the guide nucleic acid is delivered via direct uptake in the roots.
  • the guide nucleic acid is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhiz.ogenes). producing a plant with transgenic hairy roots.
  • the guide nucleic acid is injected into the roots.
  • the guide nucleic acid is produced in vitro.
  • the guide nucleic acid is methylated in vitro, such as by an RNA methylase, to promote mobility.
  • the guide nucleic acid is fused to a meristem transport segment (MTS).
  • MTS meristem transport segment
  • Delivery of the guide nucleic acid can occur through the following non-exhaustive list: through use of an RNA or DNA spray comprising the guide nucleic acid and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by injection of a composition comprising the guide nucleic acid into the stem; by direct uptake in the roots of a composition comprising the guide nucleic acid; or by biolistic transformation of roots or other tissue with circular DNA expressing the guide RNA.
  • the guide nucleic acid is transported to the meristem of the plant, and is imported into the meristem nuclei.
  • the provided methods for editing a grafted scion allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the edited genome. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on the rootstock, allowing for direct comparison of the results of providing different guide nucleic acids, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide nucleic acids, and phenotypic changes as a result of edits induced by different guide nucleic acids.
  • the provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.
  • a strain of Agrobacterium is developed that comprises the sequence specific endonuclease, and this strain can be used to infect and transform a variety of plants. This results in a variety of plants to which a guide nucleic acid can be delivered to produce heritable edits in the plant meristem. This method does not require any additional generations between the transformation with Agrobacterium and the production of heritable edits, and is thus an improvement on current editing techniques.
  • G Uptake of guide nucleic acid by roots for editing a plant with a sequence specific binding protein fused to a recombinase
  • the present application provides methods of editing a genomic target in a plant meristem comprising providing a plant comprising nucleic acid encoding a sequence specific binding protein fused to a recombinase, wherein the nucleic acid encoding a sequence specific binding protein fused to a recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the root of the plant a guide nucleic acid for the sequence specific binding protein fused to a recombinase.
  • the plant comprising the nucleic acid encoding a sequence specific binding protein fused to a recombinase is a rootstock.
  • a scion is grafted onto the rootstock.
  • the genomic editing reagents are provided to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhiz.ogenes). producing a plant with transgenic hairy roots.
  • the sequence specific binding protein fused to a recombinase is delivered to the plant root by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenesf producing a plant with transgenic hairy roots.
  • the plant provides nucleic acid encoding a sequence specific binding protein fused to a recombinase to the plant vascular system.
  • the fusion of the meristem transport segment to nucleic acid encoding the sequence specific binding protein fused to a recombinase results in the nucleic acid encoding the sequence specific binding protein fused to a recombinase being transported to cells of the meristem of the scion through the plant vascular system.
  • the nucleic acid encoding the sequence specific binding protein fused to a recombinase is transported from the rootstock to the scion through the graft junction.
  • RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported by plant vascular system.
  • RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported through the xylem or the phloem. In some embodiments, RNA encoding the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is transported to the meristem, wherein the sequence specific binding protein fused to a recombinase and/or the guide nucleic acid is translated in the meristem. Nucleic acid encoding the sequence specific binding protein fused to a recombinase is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei.
  • the guide nucleic acid is delivered to the roots. In some embodiments, the guide nucleic acid is delivered via direct uptake in the roots. In some embodiments, the guide nucleic acid is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhiz.ogenes). producing a plant with transgenic hairy roots. In some embodiments, the guide nucleic acid is injected into the roots. In some embodiments, the guide nucleic acid is produced in vitro. In some embodiments, the guide nucleic acid is methylated in vitro, such as by an RNA methylase, to promote mobility.
  • the guide nucleic acid is fused to a meristem transport segment (MTS).
  • Delivery of the guide nucleic acid can occur through the following non-exhaustive list: through use of an RNA or DNA spray comprising the guide nucleic acid and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by injection of a composition comprising the guide nucleic acid into the stem; by direct uptake in the roots of a composition comprising the guide nucleic acid; or by biolistic transformation of roots or other tissue with circular DNA expressing the guide RNA.
  • the guide nucleic acid is transported to the meristem of the plant, and is imported into the meristem nuclei.
  • the provided methods for editing a grafted scion allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the edited genome. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on the rootstock, allowing for direct comparison of the results of providing different guide nucleic acids, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide nucleic acids, and phenotypic changes as a result of edits induced by different guide nucleic acids.
  • the provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.
  • a strain of Agrobacterium is developed that comprises the sequence specific binding protein fused to a recombinase, and this strain can be used to infect and transform a variety of plants. This results in a variety of plants to which a guide nucleic acid can be delivered to produce heritable edits in the plant meristem. This method does not require any additional generations between the transformation with Agrobacterium and the production of heritable edits, and is thus an improvement on current editing techniques.
  • the method provided herein comprise editing a grafted scion. Grafting can be performed, for example, by inserting one or more cut scion stems into a cut of a rootstock stem, wherein the vascular tissue of the scion stem and the rootstock stem are substantially aligned.
  • a stabilization device may be used.
  • a successful graft exhibits a continuous vascular system from rootstock to scion, including transmission through a graft junction.
  • Sequence specific endonucleases and/or DNAs or RNAs encoding sequence specific endonucleases are expressed in the rootstock and enter the phloem and transit to the shoot apical meristem of the scion.
  • the sequence specific endonucleases and/or DNAs or RNAs encoding sequence specific endonucleases are imported into cells of the meristem.
  • sequence specific endonucleases and/or DNAs or RNAs encoding sequence specific endonucleases are able to induce targeted recombination in the genome of the meristem of the plant scion absent a guide nucleic acid.
  • sequence specific endonucleases and/or DNAs or RNAs encoding sequence specific endonucleases are processed into functional RNPs, which are able to induce targeted recombination in the genome of the meristem of the plant scion.
  • the present disclosure provides methods of editing the genome of a transgene - free plant scion, wherein the plant scion genome does not contain DNA encoding reagents for genomic modification.
  • This technology enables one to introduce constructs encoding sequence specific endonucleases into an easily transformable germplasm that can then be grafted to elite shoots as a rootstock, resulting in heritable genome edits in the scion.
  • a plant scion transformed through the present methods of genomic editing does not contain transgenes encoding the sequence specific endonucleases used to induce the targeted recombination.
  • a successful graft exhibits a continuous vascular system from rootstock to scion, including transmission through a graft junction.
  • a sequence specific binding protein fused to a recombinase and/or DNAs or RNAs encoding a sequence specific binding protein fused to a recombinase are expressed in the rootstock and enter the phloem and transit to the shoot apical meristem of the scion.
  • the sequence specific binding protein fused to a recombinase and/or DNAs or RNAs encoding a sequence specific binding protein fused to a recombinase are imported into cells of the meristem.
  • sequence specific binding protein fused to a recombinase and/or DNAs or RNAs encoding a sequence specific binding protein fused to a recombinase are able to induce targeted recombination in the genome of the meristem of the plant scion absent a guide nucleic acid.
  • sequence specific binding protein fused to a recombinase and/or DNAs or RNAs encoding a sequence specific binding protein fused to a recombinase are processed into functional RNPs, which are able to induce targeted recombination in the genome of the meristem of the plant scion.
  • the present disclosure provides methods of editing the genome of a transgene-free plant scion, wherein the plant scion genome does not contain DNA encoding reagents for genomic modification.
  • This technology enables one to introduce constructs encoding a sequence specific binding protein fused to a recombinase into an easily transformable germplasm that can then be grafted to elite shoots as a rootstock, resulting in heritable genome edits in the scion.
  • a plant scion transformed through the present methods of genomic editing does not contain transgenes encoding the sequence specific binding protein fused to a recombinase used to induce the targeted recombination.
  • a successful graft exhibits a continuous vascular system from rootstock to scion, including transmission through a graft junction.
  • Sequence specific recombinases and/or DNAs or RNAs encoding sequence specific recombinases are expressed in the rootstock and enter the phloem and transit to the shoot apical meristem of the scion.
  • the sequence specific recombinase and/or DNAs or RNAs encoding sequence specific recombinases are imported into cells of the meristem.
  • sequence specific recombinase and/or DNAs or RNAs encoding a sequence specific recombinase are able to induce targeted recombination in the genome of the meristem of the plant scion absent a guide nucleic acid.
  • sequence specific recombinase and/or DNAs or RNAs encoding a sequence specific recombinase are processed into functional RNPs, which are able to induce targeted recombination in the genome of the meristem of the plant scion.
  • the present disclosure provides methods of editing the genome of a transgene-free plant scion, wherein the plant scion genome does not contain DNA encoding reagents for genomic modification.
  • This technology enables one to introduce constructs encoding sequence specific recombinases into an easily transformable germplasm that can then be grafted to elite shoots as a rootstock, resulting in heritable genome edits in the scion.
  • a plant scion transformed through the present methods of genomic editing does not contain transgenes encoding the sequence specific recombinases used to induce the targeted recombination.
  • the plant scion must be able to be grafted onto a transformed rootstock, but it is not necessary that the plant scion itself be transformable. This widens the possibility of species that can be edited through the present disclosure. Additionally, many plants can be grafted onto the same variety of rootstock, thus speeding development of genomically edited scions.
  • the rootstock may comprise any enzyme that can induce targeted recombination and be targeted to the scion. Exemplary enzymes are detailed in the sections below.
  • the rootstock expresses a sequence specific endonuclease, such as, for example, a zinc finger nuclease (ZFN), a TALEN or a Cas protein.
  • the rootstock expresses a sequence specific binding protein, such as, for example, an inactive zinc finger nuclease (ZFN) protein, an inactive TALEN protein, a catalytically inactive Cas protein, or a transcription factor.
  • a sequence specific binding protein such as, for example, an inactive zinc finger nuclease (ZFN) protein, an inactive TALEN protein, a catalytically inactive Cas protein, or a transcription factor.
  • the sequence specific binding protein is fused to a recombinase.
  • the recombinase to which the sequence specific binding protein is fused is a Cre recombinase, a Hin recombinase, a Tre recombinase, and/or a FLP recombinase.
  • the recombinase to which the sequence specific binding protein is fused is a serine recombinase and/or a tyrosine recombinase.
  • the recombinase to which the sequence specific binding protein is fused may comprise one or more domains of a serine recombinase and/or a tyrosine recombinase, including, for example, one or more domains of a Cre recombinase, a Hin recombinase, a Tnpl recombinase, a Tre recombinase, PhiC31 integrase, a FLP recombinase, an R4 integrase, and/or a TP-901 integrase.
  • the rootstock expresses a sequence specific recombinase, such as, for example, a serine recombinase and/or a tyrosine recombinase.
  • the sequence specific endonuclease is guided to a target site in the genome of the scion by a guide RNA that is complementary to a sequence of the target site.
  • the sequence specific binding protein is fused to a recombinase is guided to a target site in the genome of the scion by a guide RNA that is complementary to a sequence of the target site.
  • the rootstock and scion are of the same species. In some embodiments, the rootstock and scion are of different species. In some embodiments, the rootstock and/or scion are from soybean, maize, or wheat. [0134] In some embodiments, to enable targeted recombination for breeding, the scions are from Fl plants. In some embodiments, the guide nucleic acids are designed to create double stranded DNA breaks where targeted recombination is preferred. In some embodiments, the methods presented herein increase the genetic gains via targeted recombination generation without additional generation time by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,
  • the methods presented herein result in doubled genetic gains via targeted recombination generation without additional generation time compared to traditional breeding.
  • any sequence-specific endonuclease that can be expressed by the rootstock and targeted to the scion may be used to induce targeted recombination in the methods of the invention.
  • the rootstock expresses a sequence specific endonuclease, such as, for example, a transcription activator-like effector nuclease (TALEN), a Cas protein, a restriction endonuclease, a meganuclease, a zinc finger nuclease (ZFN), or an Argonaute protein that is targeted to the scion.
  • TALEN transcription activator-like effector nuclease
  • Cas protein such as, for example, a transcription activator-like effector nuclease (TALEN), a Cas protein, a restriction endonuclease, a meganuclease, a zinc finger nuclease (ZFN), or an Argonaute protein that is targeted to the scion.
  • ZFN zinc finger nucle
  • the sequence specific endonuclease is guided to the target sequence in the genome of the scion by association with a guide nucleic acid, such as a guide RNA (gRNA) or a guide DNA (gDNA).
  • a guide nucleic acid such as a guide RNA (gRNA) or a guide DNA (gDNA).
  • gRNA guide RNA
  • gDNA guide DNA
  • the sequence specific endonuclease is a DNA-guided endonuclease, such as, for example, a Natronobacterium gregoryi Argonaute (NgAgo) protein (as described in Gao, F. et al. (2016) DNA-guided genome editing using the Natronobacterium gregoryi Argonaute.
  • NgAgo Natronobacterium gregoryi Argonaute
  • Argonaute proteins include Pyrococcus furiosus Argonaute (PfAgo) and Thermus thermophilus Argonaute (TtAgo).
  • sequence specific endonuclease is an RNA-guided endonuclease, such as, for example, a Cas protein.
  • RNA-guided endonuclease such as, for example, a Cas protein.
  • Cas proteins and associated CRISPR systems, ZFNs, TALENS, and meganucleases are described in more detail in below sections. J. Sequence-specific binding proteins
  • a sequence-specific binding protein may be used to target a recombinase or endonuclease to a location in the genome of the scion in order to induce targeted recombination. Any sequence- specific binding protein that can be expressed by the rootstock and targeted to the scion may be used to in the methods of the invention.
  • the rootstock expresses a sequence specific binding protein that lacks or has reduced nuclease activity, such as, for example, an inactive transcription activator-like effector nuclease (inactive TALEN), an inactive or “dead” Cas protein or other inactive sequence specific nuclease, or a transcription factor.
  • nucleases are used in combination with a sequence- specific binding protein to induce targeted recombination.
  • Non-limiting examples of other nucleases that may be expressed by the rootstock of the present invention and targeted to specific genome sequences in the scion include, for example, PvuII, Pept071, MutH, TevI, Clo051, FokI, AZwI, StsI, Mlyl, Shfl, Sdal, and CleDORF, and variants, homologs, and mutants thereof.
  • the sequence specific binding protein is guided to the target sequence in the genome of the scion by association with a guide nucleic acid, such as a guide RNA (gRNA) or a guide DNA (gDNA).
  • a guide nucleic acid such as a guide RNA (gRNA) or a guide DNA (gDNA).
  • the sequence specific binding protein is a DNA-guided endonuclease, such as, for example, an inactive Argonaute protein, or a homolog, analog, mutated, or otherwise modified version thereof.
  • the sequence specific binding protein is an RNA-guided endonuclease, such as, for example, an inactivated or “dead” Cas protein, such as, for example a dCas9.
  • the sequence specific binding protein is a transcription factor that binds to a specific sequence within the genome of the scion.
  • the transcription factor is native to the scion genome.
  • the transcription factor has been engineered to bind to a specific sequence within the genome of the scion.
  • sequence-specific recombinase that can be expressed by the rootstock and targeted to the scion may be used to induce targeted recombination in the methods of the invention.
  • the recombinase gains sequence specificity by being tethered or fused to a sequence specific binding protein, such as a sequence specific binding protein that lacks or has reduced nuclease activity, such as, for example, an inactive transcription activator-like effector nuclease (inactive TAEEN), an inactive or “dead” Cas protein or other inactive sequence specific nuclease, or a transcription factor, and that has sequence specificity for a target sequence in the genome of the scion.
  • a sequence specific binding protein such as a sequence specific binding protein that lacks or has reduced nuclease activity, such as, for example, an inactive transcription activator-like effector nuclease (inactive TAEEN), an inactive or “dead” Cas protein or other inactive sequence specific nuclease
  • the rootstock expresses a dCas9-serine recombinase fusion protein, such as recCas9 (Chaikind, B. et al. (2016) A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res. Nov 16;44(20):9758-9770) in which the dCas9 domain associates with a gRNA that is designed to bind to a target sequence in the genome of the scion and the serine recombinase domain triggers recombination, leading to site-specific recombination.
  • recCas9 Chokind, B. et al. (2016) A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res. Nov 16;44(20):9758-9770
  • the dCas9 domain associate
  • custom sequencespecific recombinases may be made and used according to the methods of the invention, including, for example by incorporating at least the recombinase domain of any type of serine or tyrosine recombinase protein (including but not limited to a serine recombinase such as Hin, Cin, or Gin recombinase) into, e.g., a fusion protein comprising a site-specific DNA binding domain.
  • the recombinase itself has sequence specificity towards a recombinase site in the genome of the scion.
  • the sequence specific recombinase may comprise one or more domains of a serine recombinase and/or a tyrosine recombinase, including, for example, one or more domains of a Cre recombinase, a Hin recombinase, a Tnpl recombinase, a Tre recombinase, PhiC31 integrase, a FLP recombinase, an R4 integrase, and/or a TP-901 integrase.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated endonucleases
  • a Cas endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” (gRNAs) that target single- or double-stranded DNA sequences.
  • gRNAs sequence-specific, non-coding “guide RNAs”
  • CRISPR loci encode both Cas endonucleases and “CRISPR arrays” of the non-coding RNA elements that determine the specificity of the CRIS PR-mediated nucleic acid cleavage.
  • the genomic DNA sequence targeted for editing or modification must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences are short and relatively non-specific, appearing throughout a given genome.
  • PAM protospacer adjacent motif
  • CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'- NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'- NGGNG (Streptococcus thermophilus CRISPR3), 5'-NNGRRT or 5'-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5'-NNNGATT (Neisseria meningitidis).
  • NGG Streptococcus pyogenes
  • 5'-NNAGAA Streptococcus thermophilus CRISPR1
  • 5'- NGGNG Streptococcus thermophilus CRISPR3
  • 5'-NNGRRT or 5'-NNGRR Spaphylococcus aureus Cas9, SaCas9
  • 5'-NNNGATT Neisseria meningitid
  • Cas9 endonucleases are associated with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleaving of the target DNA at a location three nucleotides upstream from (5' from) the PAM site.
  • Casl2a (Cpfl) CRISPR systems cleave the target DNA adjacent to a short T-rich PAM sequence, e.g., 5'-TTN, in contrast to the G-rich PAM sequences identified for Cas9 systems.
  • Examples of Casl2a PAM sequences include those for the naturally occurring Acidaminococcus sp.
  • Casl2a can also recognize a 5'-CTA PAM motif.
  • Other examples of potential Casl2a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide).
  • Various methods including in silico and/or wet lab methods) for identification of the appropriate PAM sequence and any convenient method can be used.
  • a PAM sequence can be identified using a PAM depletion assay.
  • Casl2a cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5- nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. Cell 2015, 163: 759-771. z. Cas nucleases
  • CRISPR systems Two classes (1 and 2) of CRISPR systems have been identified across a wide range of bacterial hosts.
  • the well characterized class 2 CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class 2 CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”), see Guide RNA below.
  • the Cas 12a (“Cpfl”) CRISPR system includes the type V endonuclease Cas 12a (also known as “Cpfl”).
  • Cas 12a nucleases are characterized as having only a RuvC nuclease domain, in contrast to Cas9 nucleases which have both RuvC and HNH nuclease domains.
  • Cas 12a nucleases are generally smaller proteins than Cas9 nucleases and can function with a smaller guide RNA (e.g., a crRNA having at least one spacer flanked by direct repeats), which are practical advantages in that the nuclease and guide RNAs are more economical to produce and potentially more easily delivered to a cell.
  • Casl2a nucleases examples include AsCasl2a or “AsCpfl” (from Acidaminococcus sp.) and LbCasl2a or “LbCpfl” (from Lachnospiraceae bacteria).
  • Casl2a-associated (“Cpfl ’’-associated) CRISPR arrays have been reported to be processed into mature crRNAs without the requirement of a tracrRNA, i.e., the naturally occurring Casl2a (Cpfl) CRISPR system was reported to require only the Casl2a (Cpfl) nuclease and a Casl2a crRNA to cleave the target DNA sequence; see Zetsche et al. Cell 2015, 163: 759-771; U.S. Pat. No. 9,790,490.
  • nuclease activity for cutting DNA followed by repair by the endogenous cell machinery is one solution to generate useful mutants.
  • the nuclease activity can be eliminated or altered, as in dCas (“dead” Cas, i.e., Cas with no nuclease functionality but retains DNA binding capabilities) or nCas (“nickase” Cas, i.e., Cas that makes single- stranded breaks rather than double- stranded breaks but retains DNA binding capabilities), TALE (TAL-effector), or ZF (zinc finger) versions of the polypeptides.
  • dead Cas i.e., Cas with no nuclease functionality but retains DNA binding capabilities
  • nCas nickase” Cas, i.e., Cas that makes single- stranded breaks rather than double- stranded breaks but retains DNA binding capabilities
  • TALE TAL-effector
  • ZF zinc finger
  • Inactivated nucleases can be useful for targeting the desired DNA sequence, while editing can be performed by nucleobase editors attached to the altered nucleases. Examples are included in W02018176009 and US Patent No. 10,113,163, incorporated herein by reference.
  • CRISPR-based RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9 (also known as Csnl and Csxl2), Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337).
  • Cas 12 is used herein to refer to any Cas 12 protein, including but not limited to Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337.
  • the Cas nuclease is selected from the group consisting of Cas9, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4, C2cl, C2c2, C2
  • the Cas nuclease is a Cas nickase. In some embodiments, the Cas nuclease is a Cas9 nickase or a Cas 12 nuclease. In some embodiments, the Cas nickase is a Cas9 nickase or a Casl2 nickase. In some embodiments, the Cas nickase comprises mutation in one or more nuclease active sites. In some embodiments, the Cas nuclease is associated with a reverse transcriptase.
  • An exemplary SpCas9 amino acid sequence (UniProt primary accession no. Q99ZW2) is presented in SEQ ID NO: 63:
  • a Cas nuclease is encoded by a nucleic acid.
  • the nucleic acid encoding the Cas nuclease is codon-optimized for use in a species of plant.
  • the Cas nuclease is codon-optimized for expression in dicots.
  • the Cas nuclease is codon-optimized for expression in soybean.
  • the Cas nuclease is codon-optimized for expression in monocots.
  • the Cas nuclease is codon-optimized for expression in com.
  • the Cas nuclease is codon-optimized for expression in wheat.
  • the Cas nuclease is fused to a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • CRISPR nuclease fusion proteins containing nuclear localization signals and codon-optimized for expression in maize are disclosed in U.S. patent application Ser. No. 15/120,110, published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to PCT/US2015/018104 (published as WO/2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700), incorporated herein by reference.
  • the nucleic acid encoding the Cas nuclease is fused to a nucleic acid encoding a meristem transport segment (MTS).
  • MTS meristem transport segment
  • the nucleic acid encoding at least one guide RNA and the nucleic acid encoding the Cas nuclease are fused to one or more nucleic acids encoding a meristem transport segment.
  • RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion by the plant vascular system.
  • RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion through the xylem or the phloem.
  • RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion through the plasmodesmata. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are translated in the cytosol of a meristem cell. In some embodiments, translation of the RNA encoding the Cas nuclease and at least one guide RNA in the cytosol of a meristem cell results in editing of the genome of the meristem cell. In some embodiments, the meristem is on the plant scion. [0149] In some embodiments, the nucleic acid encoding the Cas enzyme is operably linked to a promoter.
  • useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).
  • the nucleic acid encoding the Cas enzyme is operably linked to a constitutive promoter.
  • constitutive promoters include a CaMV 35S promoter as disclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Pat. No.
  • the nucleic acid encoding the Cas enzyme is operably linked to an inducible promoter.
  • An “inducible” promoter is a promoter that initiates transcription in response to an environmental stimulus such as heat, cold, drought, light, or other stimuli, such as wounding or chemical application. Examples of inducible promoters include, but are not limited to, those described in U.S. Pat. No. 6,294,714 (light inducible promoters), U.S. Pat. No.
  • the nucleic acid encoding the Cas enzyme is operably linked to a promoter selected from the group consisting of promoters active in roots and promoter active in phloem companion cells.
  • the promoter active in roots is the promoter of a gene selected from the group consisting of Arabidopsis thaliana WRKY6 or orthologous genes thereof, chickpea WRKY31 or orthologous genes thereof, carrot MYB113 or orthologous genes thereof, corn GLU1 or orthologous genes thereof, strawberry RB7-type TIP-2 or orthologous genes thereof, and banana TIP2-2 or orthologous genes thereof.
  • Additional suitable root promoters are provided in the RGPDB database (database of root-associated genes and promoters in maize, soybean, and sorghum) as described in Moisseyev et al. Database, 1-7 (2020).
  • the promoter active in phloem companion cells is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FORI gene (Noll et al. Plant Mol Biol 2007, 65(3): 285-294), a rice tungro bacilliform virus promoter (Yin et al.
  • JMJ18 JmjC domain-containing protein 18
  • PP2 phloem protein 2
  • the nucleic acid encoding the Cas nuclease and/or at least one guide RNA is intended to be transcribed in the rootstock.
  • the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS).
  • the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease.
  • the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the Cas nuclease.
  • the nucleic acid encoding the Cas nuclease and/or guide RNA is intended to be transcribed in a cell of the rootstock, transported through the graft junction to the scion, and translated inside a scion meristem cell.
  • RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system.
  • RNA encoding the Cas nuclease and/or the guide RNA is transported from the rootstock to the scion by plant vascular system.
  • RNA encoding the Cas nuclease and/or the guide RNA is transported through the xylem or the phloem.
  • RNA encoding the Cas nuclease and/or the guide RNA is transported to the scion through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is translated in the scion. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported to the meristem, wherein the Cas nuclease and/or the guide RNA is translated in the meristem. As such, the nucleic acid encoding the Cas nuclease and/or the guide RNA is typically embedded within an mRNA component.
  • a 5’ cap and polyA tail are also useful in stabilizing the RNA.
  • a 5’ UTR has translation initiation sequences upstream of the Cas coding sequence.
  • a 5’ UTR can also have small upstream open reading frames that affect translation (Jorgensen and Dorantes- Acosta, Front. Plant Sci 2012, 3:191).
  • an mRNA can comprise a 5’ UTR comprising a 7-methylguanosine cap at its 5’ terminus followed by an untranslated sequence and terminated by the translation initiation codon of the coding sequence (e.g., the Cas coding sequence).
  • the nucleic acid encoding the Cas nuclease can be optimized to increase nuclease activity and editing efficiency.
  • the nucleic acid encoding the Cas enzyme is operably linked to a nuclear localization signal (NLS), such as the NLS from SV40.
  • NLSs nuclear localization signal
  • the nucleic acid encoding the Cas nuclease is fused to a cell penetrating peptide (CPP), such as octa-arginine or nona-arginine or a homoarginine 12-mer oligopeptide, or a CPP disclosed in the database of cell-penetrating peptides CPPsite 2.0, publicly available at webs[dot]iiitd[dot]edu[dot]in/raghava/cppsite/ (Kardani and Bolhassani J Mol Biol 2021, 433(11): 166703).
  • the nucleic acid encoding the Cas enzyme further comprises a terminator.
  • terminal is meant a DNA segment near the 3' end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA.
  • a 3' element is also sometimes referred to as a “3 '-untranslated region” or “3'-UTR” or a “polyadenylation signal”.
  • Non-limiting embodiments of terminators functional in eukaryotic cells include a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3' element from an Agrobacterium sp.
  • 3' elements include: Agrobacterium tumefaciens nos 3', tml 3', tmr 3', tins 3', ocs 3', and tr7 3' elements disclosed in U.S. Pat. No.
  • the terminator is selected from the group consisting of CaMV 35S terminator, Atug7 terminator, NOS terminator, Act2 terminator, MAS terminator, tomato ATPase terminator, rbcSC3 terminator, potato H4 terminator, rbcSE9 terminator, GILT terminator, ALB terminator, API terminator, HSP terminator, and OCS terminator , as referenced in Hassan et al. (Trends Plant Sci 2021, 26: 1133-1152).
  • the nucleic acid encoding the Cas enzyme further comprises one or more introns.
  • the nucleic acid encoding the Cas enzyme further comprises one or more transcriptional enhancers.
  • the one or more transcriptional enhancers comprise one or more bacterial octopine synthase (OCS) enhancers (U.S. Patent No. 11,198,885).
  • OCS bacterial octopine synthase
  • the nucleic acid encoding the Cas enzyme further comprises a triple OCS enhancer (U.S. Patent No. 11,198,885).
  • the nucleic acid encoding the Cas enzyme further comprises a 5’ UTR comprising a translational enhancer.
  • the nucleic acid encoding the Cas enzyme further comprises a Kozak sequence endogenous to the scion species at the translation start codon.
  • the nucleic acid encoding the Cas enzyme further comprises nuclear localization signals flanking the coding sequence of the Cas enzyme. ii. Guide RN As
  • CRISPR-based RNA-guided nuclease systems typically require an effector polypeptide and one or more guide RNAs (gRNAs).
  • the guide RNAs are generally made up of an effector-binding region and a target DNA recognition region, and in some embodiments include tracrRNAs.
  • a “trans-activating crRNA” or “tracrRNA” is a trans-encoded small RNA that is partially homologous to repeats within a CRISPR array. At least in the case of Cas9 type CRISPR systems, both a tracrRNA and a crRNA are required for the CRISPR array to be processed and for the nuclease to cleave the target DNA sequence.
  • the Cas9 crRNA contains a “spacer sequence”, typically an RNA sequence of about 20 nucleotides (in various embodiments this is 20, 21, 22, 23, 24, 25, or up to about 30 contiguous nucleotides in length) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence of about equivalent length.
  • the Cas9 crRNA also contains a region that binds to the Cas9 tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA:tracrRNA hybrid or duplex.
  • the crRNA:tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence; in some examples, a tracrRNA and crRNA (e.g., a crRNA including a spacer sequence) can be included in a chimeric nucleic acid referred to as a “single guide RNA” (sgRNA).
  • sgRNA single guide RNA
  • guide RNA refers to a nucleic acid that comprises or includes a nucleotide sequence (sometimes referred to a “spacer sequence”) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence (e.g., a contiguous nucleotide sequence that is to be modified) in a genome; the guide RNA functions in part to direct the CRISPR nuclease to a specific location on the genome.
  • a gRNA is a CRISPR RNA (“crRNA”), such as the engineered Casl2a crRNAs described in this disclosure.
  • the gRNA can be a tracrRNA:crRNA hybrid or duplex, or can be provided as a single guide RNA (sgRNA).
  • At least 16 or 17 nucleotides of gRNA sequence corresponding to a target DNA sequence are required by Cas9 for DNA cleavage to occur; for Casl2a (Cpfl) at least 16 nucleotides of gRNA sequence corresponding to a target DNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence corresponding to a target DNA sequence were reported necessary for efficient DNA cleavage in vitro; see Zetsche et al. Cell 2015, 163: 759-771.
  • Casl2a (Cpfl) endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S. Pat. No.
  • guide RNA sequences are generally designed to contain a spacer sequence of between 17-24 contiguous nucleotides (frequently 19, 20, or 21 nucleotides) with exact complementarity (e.g., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having spacers with less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a spacer having a length of 20 nucleotides and between 1-4 mismatches to the target sequence), but this can increase the potential for off-target effects.
  • Guide RNA(s) can be part of the same RNA (mRNA) capable of expressing the Cas nuclease.
  • one or more guide RNAs are flanked by direct repeats (DR) of the CRISPR array from which the Cas effector polypeptide was first isolated.
  • the two or more guide RNAs are each flanked by a direct repeat.
  • a translated and expressed active Cas 12a nuclease can process the DR-flanked spacers of the mRNA to make guide RNAs.
  • a translated and expressed active Casl2a nuclease can process Casl2a DR-flanked spacers of the mRNA to make guide RNAs.
  • a translated and expressed active Casl2e nuclease can process Casl2e DR-flanked spacers of the mRNA to make guide RNAs.
  • a translated and expressed active Casl2i nuclease can process Casl2i DR- flanked spacers of the mRNA to make guide RNAs.
  • a translated and expressed active Casl2j nuclease can process Casl2j DR- flanked spacers of the mRNA to make guide RNAs.
  • a guide RNA suitable for matching an expressed effector polypeptide is flanked by processing elements, so that functional guide RNAs are excised inside the cells.
  • Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs (see Mikami et al. Plant Cell Physiol. 2017, 58(11): 1857-1867; and US Patent No. 10,308,947).
  • Ribozymes can autocatalytically cleave the RNA to release the guide RNA from a polycistronic transcript and/or remove additional 5’ or 3’ sequence around the guide RNA.
  • tRNAs are processed by elements of the cell’s endogenous tRNA system, such as RNase P, RNase Z, and RNase E, and tRNA sequences or pre-tRNA sequences can also be used to release a guide RNA flanked by processing elements from a polycistronic transcript and/or remove additional 5’ or 3’ sequence around the guide RNA.
  • the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences.
  • each of the ribozyme sequences is independently selected from the group consisting of a hammerhead ribozyme sequence, a HDV ribozyme sequence, a Csy4 sequence, and a tRNA processing enzyme sequence.
  • the nucleic acid encoding the guide RNA and the MTS further comprises a hammerhead ribozyme sequence 5’ to the nucleic acid encoding the guide RNA and the MTS, and a HDV ribozyme 3’ to the nucleic acid encoding the guide RNA and the MTS.
  • a guide RNA is encoded by a nucleic acid.
  • the guide RNA is fused to a meristem transport segment (MTS).
  • the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas9 nickase or Casl2 nuclease and/or 3’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the Cas9 nickase or Casl2 nuclease and/or 5’ of the nucleic acid encoding the guide RNA.
  • the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target.
  • the nucleic acid encoding the guide RNA and the MTS further comprises a terminator.
  • the terminator is a U6 terminator.
  • the guide RNA comprises a 5-methycytosine group.
  • the present invention comprises a guide RNA or guide RNA(s) which have chemical modifications. Chemical modifications are made to RNA molecules which then alter at least one of the four canonical ribonucleotides: A, U, C, and G. These modifications can be natural or unnatural and refer to a chemical moiety or portions of a chemical moiety which are not found in the unmodified canonical ribonucleotides.
  • Alternative bases can include but are not limited to 2-thiouridine, 4-thiorudine, 2- aminoadenosine, 7-deazaguanosine, inosine, 5-methylcytidine, 5-aminoallyluridine, and 5- methyluridine.
  • a guide RNA which comprises any backbone or inter-nucleotide linkage other than a natural phosphodiester linkage is a chemically modified guide RNA.
  • Alternative phosphodiester linkages can include but are not limited to an alkylphosphonate, a phosphonocaboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phoshporodithioate linkage.
  • a guide RNA which comprises labeled isotopes such as one or more of 15 N, 13 C, 14 C, Deuterium, or 32 P, or other atoms used as tracers, is a modified guide RNA.
  • a guide RNA which comprises modifications made to the sugar group is a chemically modified RNA.
  • Sugar group modifications can include but are not limited to 2’-O-methyl, 2’ -deoxy, 2’ -methoxyethyl, 2’fluoro, 2’-amino, a sugar in L form, and 4’-thioribosyl.
  • chemical modifications protect the guide RNA from nucleases. In certain embodiments, this modification aids in the stability of the RNA molecules, where the half-life of the chemically modified RNA molecule is altered from the unmodified form.
  • the chemically modified guide RNA maintains its functionality, which includes guide RNA binding to a Cas protein. In some embodiments, this maintained functionality of the gRNA includes binding a target polynucleotide.
  • the maintained functionality of the guide RNA includes binding both a Cas protein and a polynucleotide in complex.
  • the chemical modifications on the guide RNA are used to distinguish the sequences from the nascent sequences present in the experimental plant.
  • the chemical modifications alter the prevalence of off-target cleavage events, where “off-target” is defined as a site in the target genome that is different from the site at which the guide RNA was designed to induce a cleavage event.
  • the guide RNA further comprises (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide RNA; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide RNA; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar.
  • each of the one or more modified nucleotides is independently selected from the group consisting of a 2'-O-methyl nucleotide, a 2'-O-methyl-3'-phosphorothioate nucleotide, a 2'-O-methyl-3'-phosphonoacetate nucleotide, and a 2'-O-methyl-3'- phosphonothioacetate nucleotide.
  • the one or more modified nucleotide comprises a modified intemucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group.
  • the nucleic acid encoding the guide RNA is operably linked to a promoter.
  • the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.
  • the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the rootstock.
  • a single guide RNA is provided to the plant.
  • multiple guide RNAs are provided to the plant.
  • the multiple guide RNAs are provided in an array.
  • multiple guide RNAs are provided in an array in order to facilitate multiplexed editing.
  • the two or more guide RNAs are encoded by a single precursor RNA.
  • guide arrays can be designed to contain one or multiple guide RNAs designed to target a DNA sequence for editing, where the guide RNA includes at least one spacer sequence that corresponds to a specific locus of about equivalent length in the target DNA; see, for example, Cong et al. Science, 2013, 339: 819-823; Ran et al. Nature Protocols, 2013, 8: 2281-2308.
  • the guide arrays contain multiple guide nucleic acids that are designed to target multiple different DNA sequence for editing.
  • a guide array may comprise one or more different guide nucleic acids that target a first DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a second DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a third DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a fourth DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a fifth DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a sixth DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a seventh DNA sequence in the genome of the scion, one or more different guide nucleic acids that target an eighth DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a ninth DNA sequence in the genome of the scion, one or more different guide nucleic acids that target a tenth DNA
  • multiple guide nucleic acids may have overlapping target sequences. In some embodiments, multiple guide nucleic acids may comprise non-identical sequences but target the same sequence. In some embodiments, different guide nucleic acids may target the same or overlapping target sequences with varying affinities. In some embodiments, different guide nucleic acids may have target sequences that are complementary or partially complementary to one another. In some embodiments, one or more guide nucleic acids may target one or more variants of a gene present in the genome or gene pool of the scion.
  • the gRNA array comprises more than one spacer sequence. In some embodiments, the gRNA array comprises more than one distinct spacer sequences. In some embodiments, the gRNA array comprises more than one distinct spacer sequences designed to target the same genomic locus. In some embodiments, the gRNA array comprises more than one distinct spacer sequences designed to target more than one distinct genomic loci.
  • the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are operably linked to a single promoter. In other embodiments, the multiple guide RNAs are operable linked to multiple promoters. In some embodiments, the multiple guide RNAs are operably linked to multiple copies of the same promoter.
  • the multiple guide RNAs are operably linked to different promoters. In some embodiments, the multiple guide RNAs target the same genomic locus. In other embodiments, the multiple guide RNAs target multiple genomic loci. In some embodiments, the multiple guide RNAs are provided in a gRNA array, wherein the gRNA array is operably linked to a single MTS. In some embodiments, the method comprises applying two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each joined to an MTS.
  • the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are operably linked to a single meristem transport segment (MTS). In other embodiments, the multiple guide RNAs are operable linked to multiple MTSs. In some embodiments, the multiple guide RNAs are operably linked to multiple copies of the same MTS. In some embodiments, the multiple guide RNAs are operably linked to different MTSs.
  • MTS meristem transport segment
  • delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem.
  • the composition comprising the guide RNA comprises a surfactant.
  • the composition comprising the guide RNA comprises glass beads coated with the guide RNA.
  • delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem.
  • delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem.
  • delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf.
  • the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • the guide RNA is delivered to the plant root by incubating the root with a composition comprising the guide RNA.
  • the guide RNA is delivered to the plant root by an Agrobacterium rhizogenes transformation. In some embodiments, the Agrobacterium rhizogenes transformation produces transgenic hairy roots.
  • the guide RNA is delivered to the plant root by injecting a composition comprising the guide RNA into the root.
  • the composition comprising the guide RNA comprises a nuclease inhibitor, optionally, wherein the nuclease inhibitor is an RNase inhibitor.
  • the composition comprising the guide RNA comprises a nuclease inhibitor.
  • the nuclease inhibitor comprises an RNase inhibitor.
  • application comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, stem, and/or meristem.
  • biolistic transformation comprises transformation of circular DNA encoding the guide RNA.
  • Zinc finger nucleases are synthetic proteins comprising a non-specific cleavage domain from the FokI restriction endonuclease fused to a zinc finger DNA-binding domain that has been engineered to be specific to a desired DNA sequence, where the active ZFN is in the form of a dimer of two monomers, which each comprise the cleavage domain and the DNA binding domain.
  • ZFNs can be engineered to cleave almost any long stretch of double-stranded DNA, enabling customization for nearly any genetic sequence.
  • the sequence-specific DNA-binding domain in a ZFN usually comprises 3 or 4 zinc finger arrays. Relative to the start of the zinc finger co-helix, the amino acids at positions -1, +2, +3, and +6 determine site specificity, and are engineered using various methods (such as, e.g., Context-dependent Assembly (CoDA), Modular Assembly, and Oligomerized Pool Engineering (OPEN)) to bind to a desired target DNA sequence. The remaining amino acids of the DNA binding domain are conserved and make up the backbone of the domain.
  • CoDA Context-dependent Assembly
  • OFPEN Oligomerized Pool Engineering
  • DNA cleavage is mediated by the FokI nuclease domain, which must be present as a dimer in order to cleave DNA.
  • an active ZFN dimer requires two ZFNs monomers with palindromic binding sites such that each can bind complementary strands of DNA in the double-stranded cut site.
  • the C-terminal regions of the monomers each bind the cleavage site (guided by the DNA binding domain) on opposite strands of DNA, offset by about 5 to 7 base pairs. The ZFN then cleaves the target site.
  • ZFN is used broadly herein and can refer to one or both members of a ZFN dimer as described above, as well as to, e.g., a monomeric ZFN that can cleave double stranded DNA without association with another ZFN.
  • Transcription activator-like effectors are site specific DNA binding domains that can be engineered to specifically bind nearly any DNA sequence, which, when combined with a nuclease (N) domain (or, alternatively, with a different functional cleavage domain, such as, e.g., a recombinase, or a transposase), then form a TALEN protein with sequence specific endonuclease activity.
  • N nuclease
  • a different functional cleavage domain such as, e.g., a recombinase, or a transposase
  • the TALE moves to the plant cell nucleus, where it recognizes and binds to a specific DNA sequence within a promoter a specific host gene.
  • Each TALE comprises a central DNA-binding domain containing 13-28 repeat monomers, which each contain 33-34 amino acids that are highly conserved except for at positions 12 and 13.
  • the residues at positions 12 and 13 are hypervariable and are known as repeat-variable diresidues (RVDs).
  • RVDs repeat-variable diresidues
  • These may be engineered using methods such as, e.g., software programs (e.g., DNA Works); Doyle et al. (2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res.
  • TALEs can be engineered to contain various combinations of repeat segments with RVDs chosen to target a specific sequence of interest.
  • TALEN any DNA cleavage domain, including but not limited to those described herein, may be fused to a TALE domain to function as a TALEN.
  • the term TALEN is used broadly herein and can refer to, e.g., TALEN monomers, dimers (such as, for example, a dimers of monomers each comprising a FokI sequence is fused to a TALE, wherein the site specificities of the TALEs flank the target cleavage site, such that the FokI monomers dimerize and create a double strand break at the target site), or multimers.
  • Meganucleases are unique, highly active enzymes that recognize and digest sitespecific DNA targets of about 14 base pairs and longer. Meganucleases occur naturally in many microbial organisms, and can be engineered for specific DNA recognition sequences, usually of about 14-40 base pairs, for example, using methods such as, for example, mutagenesis and high-throughput screening. Unlike ZFN, TALEN, and CRISPR systems, DNA recognition and cleavage are both controlled from a single domain in meganucleases.
  • the methods provided herein involve transport of one or more components of a gene editing system (e.g., any sequence specific endonuclease disclosed herein, any sequence specific binding protein fused to a recombinase disclosed herein, any sequence specific recombinase disclosed herein, and optionally further including one or more guide nucleic acids) to the meristem.
  • a gene editing system e.g., any sequence specific endonuclease disclosed herein, any sequence specific binding protein fused to a recombinase disclosed herein, any sequence specific recombinase disclosed herein, and optionally further including one or more guide nucleic acids
  • Meristem transport segments travel through the plant, typically but not limited to via the phloem, and are taken up into meristematic tissues.
  • the examples below are sequences from individual species, which sometimes work across species.
  • Arabidopsis FT-based vectors work in Nicotiana benthamiana and Arabidopsis.
  • the transport segment is based on a plant-transported RNA
  • its actual sequence may be a fragment determined by characterizing a deletion series to make a smaller sequence retaining the desired transport (phloem mobility and/or meristem cell translocation) capabilities.
  • the initiator methionine codon or translation initiation codon of the base sequence may also be mutated in some cases.
  • the Flowering Focus T (FT) mRNA is useful as a meristem transport segment.
  • SEQ ID NO: 2 shows the DNA sequence that encodes the Arabidopsis FT RNA
  • SEQ ID NO: 1 is a fraction of SEQ ID NO: 2 that encodes the RNA that functions as a transport segment.
  • Alternative useful FTs may be ZCN8 (encoded by SEQ ID NO: 3), which may work across related monocot species.
  • Alternative useful FTs may be GmFT2a (e.g., SEQ ID NO: 4; Sun et al. PEoS One. 2011, 6(12): e29238. doi:10.1371/journal.pone.0029238; Jiang et al. BMC Genomics.
  • FT RNA molecules that can be used include: (i) RNAs set forth in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (iii) FT RNAs from various plants set forth in US 20190300890, which is incorporated herein by reference in its entirety, allelic variants thereof, and meristem transport-competent (MTC) orthologs thereof, MTC variants thereof, and/or MTC fragments thereof; and tRNA-like sequences (TLSs) (Zhang et al.
  • MTC
  • FT RNA molecules that can be used include RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or a meristem transport-competent (MTC) fragment thereof.
  • MTC meristem transport-competent
  • viral and cellular-derived RNA molecules that are useful as part of a transport segment include the mRNAs of FT, GAI, CmNACP, tomato EeT6, a KNOX gene, BEE5, or tRNA-like sequences (Ruiz-Medrano et al. Development 1999, 126: 4405-4419; Kim et al. Science 2001, 293: 287-289; Haywood et al. Plant J. 2005, 42: 49-68; and Ei et al. Sci. Rep. 2011, 1: 73; Cho et al. J. Exp. Bot 2015, 66: 6835-6847; Zhang et al. Plant Cell 2016, 28: 1237-1249; and WO2017178633).
  • RNAs that can be used include: (i) RNAs set forth in SEQ ID NO: 26, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 26, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; and (iii) RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 26, or a meristem transport-competent (MTC) fragment thereof.
  • CmNACP RNAs that can be used include: (i) RNAs set forth in SEQ ID NO: 25, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 25, a MTC variant thereof, and/or a MTC fragment thereof; and (iii) RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 25, or a meristem transport-competent (MTC) fragment thereof.
  • MTC meristem transport-competent
  • LeT6 RNAs that can be used include: (i) RNAs set forth in SEQ ID NO: 27, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 27, a MTC variant thereof, and/or a MTC fragment thereof; and (iii) RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 27, or a meristem transport-competent (MTC) fragment thereof.
  • MTC meristem transport-competent
  • BEL5 RNAs that can be used include: (i) RNAs set forth in SEQ ID NO: 28, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 28, a MTC variant thereof, and/or a MTC fragment thereof; and (iii) RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 28, or a meristem transport-competent (MTC) fragment thereof.
  • MTC meristem transport-competent
  • tRNA-like RNAs examples include: (i) RNAs set forth in SEQ ID NO: 29, 30, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or a MTC fragment thereof; (ii) allelic variants of SEQ ID NO: 29, 30, a MTC variant thereof, and/or a MTC fragment thereof; and (iii) RNAs having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 29, 30, or a meristem transport-competent (MTC) fragment thereof.
  • MTC meristem transport-competent
  • a TLS sequence, SEQ ID NO: 29 or 30, a meristem transport-competent (MTC) ortholog thereof, a MTC variant thereof, and/or an MTC fragment thereof can comprise an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.
  • TLS sequences suitable for RNA transport and the structural features of such RNAs are set forth in Zhang et al. Plant Cell. 2016 Jun. 28(6): 1237, doi.org/10.1105/tpc.15.01056. [0183] Further description of biological sequences provided in the sequence listing is set forth in Table 1.
  • RNA molecules set forth in SEQ ID NO: 9-30 are respectively encoded by the DNA molecules set forth in SEQ ID NO: 31-52.
  • the meristem transport-competence (MTC) potential can be determined for any variants, fragments, and/or orthologs of the aforementioned FT, GAI, CmNACP, LeT6 a tomato KNOX gene, BEL5, or tRNA-like RNAs.
  • a side-by-side comparison with a known MTS as a positive control is useful. As such, a number of configurations can be used.
  • One approach is to fuse candidate sequences to guide sequences of characterized editing potential for a species of interest.
  • RNA sequences can be introduced into the phloem of an individual plant that expresses or translates at least in the meristem a nuclease capable of associating with the guide sequence and producing the intended genomic alteration.
  • RNA sequences can be expressed in vitro and introduced into the phloem as purified molecules.
  • a concentrated solution of RNA molecules of interest can be applied to a mechanically injured plant tissue, such as a cut or abraded leaf, stem, or meristem dome.
  • RNAs can be coated on particles, such as micro or nano-scale particles such as gold or tungsten, for biolistic delivery.
  • the RNA sequences could be incorporated into RNA viruses introduced in the plants (Jackson et al. Front. Plant Sci. 2012, 3: 127; Ali et al. Mol. Plant 2015, 8: 1288-1291; Cody et al. Plant Physiol. 2017, 175: 23-35; Ali et al. Virus Res.
  • RNAs by grafting can be assayed by introducing RNAs by grafting, i.e. the RNA molecules can be expressed in the rootstock of a grafted plant, and their effect observed in the scion (Zhang et al. Plant Cell, 2016, 28: 1237- 1249; Huang et al. Plant Physiol. 2018, 178:783-794).
  • MTS candidates can be assayed for longer and/or more complex RNA molecules, or mixtures of RNA molecules, that comprise not only guide or processable guide regions, but also nuclease-encoding sequences.
  • a clear readout of MTC is detection of the expected genomic alterations in progeny plants, which can be done by sequencing of the target genomic region, or even by whole genome sequencing. But alternative readouts can be designed that may be more convenient in some cases.
  • the guide sequences may be directed to disrupt or repair a reporter gene, such as a transgene encoding a fluorescent polypeptide.
  • the expected genetic changes can then be evaluated in the treated plants by measuring changes in the reporter.
  • Another convenient genomic alteration target in many species is phytoene desaturase (PDS), with the albino phenotype of the mutant serving as a readout.
  • PDS phytoene desaturase
  • the meristem transport segment comprises a Flowering Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.
  • the FT-derived sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.
  • the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the sequence specific endonuclease, binding protein, or recombinase and/or 3’ of the nucleic acid encoding the guide nucleic acid. In some embodiments, the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the sequence specific endonuclease, binding protein, or recombinase and/or 5’ of the nucleic acid encoding the guide nucleic acid. [0187] In some embodiments, the plant further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.
  • Embodiments of the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in editing or effecting a sequence-specific modification of a target DNA sequence or target gene in a DNA molecule, a chromosome, or a genome, wherein the edit or sequence- specific modification comprises one or more targeted recombination events in the genome of a scion.
  • the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), noncoding sequence, or both coding and non-coding sequence.
  • the methods provided herein comprise producing a targeted recombination event that brings one or more polymorphisms from a chromosome derived from a first variety into a homologous chromosome derived from a second variety.
  • the first variety is the male parent and the second variety is the female parent.
  • the first variety is the female parent, and the second variety is the male parent.
  • the homologous chromosome derived from the second variety did not previously contain the one or more polymorphisms.
  • the first variety is a breeding line.
  • the second variety is a breeding line.
  • the first and second varieties are both breeding lines.
  • the first and/or second varieties are elite breeding lines.
  • DNA sequence targets there are numerous plant-endogenous targets (i.e., DNA sequence targets) that can be used in the present methods.
  • the methods presented here can be applied to one or more genomic regions selected independently from the group consisting of a gene, an array of tandemly duplicated genes, a multigene family, an enhancer, a suppressor, a transcription factor binding site, a protein binding site, a promoter, a termination sequence, a splice acceptor sequence, a splice donor sequence, an intron, an exon, an siRNA, a sequence encoding a non-coding RNA, a microRNA, a transgene, an intergenic region, a genic region, a heterochromatic region, a euchromatic region, a region of methylated DNA, and a quantitative trait locus (QTL).
  • QTL quantitative trait locus
  • the method of the present invention may be used to introduce edits to affect any phenotype, quality, or trait of the organism.
  • the methods herein may be used to introduce edits to the genome that affect yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, or disease resistance of a plant.
  • the scion is not of a uniform line.
  • the scion is of a line that exhibits variation in trait expression when selfed or propagated. In some embodiments, the variation may be unpredictable.
  • the scion is not of an inbred line.
  • the scion is from an Fl plant.
  • the scion is from an F2 plant.
  • the scion is from an F3 plant.
  • the scion is from an F4 plant or a further generation plant.
  • the scion is from a plant produced by a cross of two different breeding lines.
  • the scion is from a plant derived from a plant produced by a cross of two different breeding lines. In some embodiments, one or both breeding lines are elite breeding lines. In some embodiments, the scion is from a plant produced by a cross of one breeding line and one wild variety. In some embodiments, the scion is from a plant produced by a cross between one breeding line and one variety that is not a breeding line.
  • a target sequence is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 150 bp of the 5’ end of a gene of interest. In some embodiments, a target sequence is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 150 bp of the 3’ end of a gene of interest. In some embodiments, a target sequence is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 150 bp of a polymorphism of interest.
  • the target sequence is within a genomic region that has sufficient surrounding 5’ and 3’ identity to allow for recombination.
  • recombination can occur across a conserved region shared by two genomic loci, such as across a conserved region between two sets of parental chromosomes, between two homologous chromosomes, between non- homologous chromosomes sharing some degree of sequence identity, or between homoeologous chromosomes.
  • some plants, such as maize are known to comprise chromosomal segments that are recalcitrant to recombination, due to, for instance, the presence of chromosomal inversions and other polymorphisms that prevent alignment of specific regions of homologous chromosomes.
  • the 5’ and/or 3’ sequences of potential target sequences are analyzed to determine whether they are likely to have sufficient sequence identity to allow for recombination.
  • the 5’ sequences of potential target sequences share sequence homology for at least the first 10, 15, 20, 25, 30, 45, or 50 bases immediately 5’ of the target sequences and the 3’ sequences of potential target sequences share sequence homology for at least the first 10, 15, 20, 25, 30, 45, or 50 bases immediately 3’ of the target sequences.
  • the 5’ sequences of potential target sequences share sequence homology for at least the first 9 bases immediately 5’ of the target sequences and the 3’ sequences of potential target sequences share sequence homology for at least the first 9 bases immediately 3’ of the target sequences (see, e.g., Fujimoto, R., et al. (2009) Minimum Length of Homology Arms Required for Effective Red/ET Recombination, Bioscience, Biotechnology, and Biochemistry, 73:12, 2783-2786, DOI: 10.1271/bbb.90584).
  • the 5’ and/or 3’ sequences of potential target sequences have at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.
  • the methods presented here can be applied to a promoter bashing or fine-tuning approach, to create a range of phenotypes based on promoter alterations of a gene of a certain sequence or gene of interest (Rodriguez-Leal et al. Cell 2017, 171(2): 470-480).
  • a target gene may be selected that has a current, baseline level of expression in a target plant species.
  • Targeted recombination events may be produced at different regions of the promoter of this target gene to incorporate, for instance, different polymorphisms.
  • Multiple lines of the elite germplasm may be generated containing distinct edits in the target gene promoter using the methods provided herein.
  • one line may have deleted a transcription factor binding site; a second line may have introduced, via recombination from a homologous chromosome, a single base pair substitution in the transcription factor binding site; a third line may have introduced two base pair substitutions in the transcription factor binding site.
  • the differentially edited promoters can be assessed for phenotype, including sub-organismal level phenotype such as RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, or translational efficiency, and/or organismal level phenotype such as yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, disease resistance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content.
  • sub-organismal level phenotype such as RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, or
  • the edit results in increased transcription compared to the baseline level of expression in a target plant species. In some embodiments, the edit results in decreased transcription compared to the baseline level of expression in a target plant species.
  • the optimal allele may be selected based on sub-organismal phenotype and/or organismal phenotype.
  • a target to be modified is a genetic variant that is known to be deleterious.
  • a target to be modified is identified by a linkage study or an association study, such as a genome-wide association study (GWAS) or a transcriptome-wide association study (TWAS).
  • GWAS genome-wide association study
  • TWAS transcriptome-wide association study
  • a target to be modified is identified through the use of statistical models, machine learning, or artificial intelligence. Deleterious genetic variants may be identified through analysis of factors including, but not limited to, evolutionary conservation (See e.g.
  • RNA expression level RNA expression level
  • gene transcript splicing ratio ribosomal occupancy
  • allele specific expression metabolite abundance
  • protein modifications micro RNA or small RNA abundance
  • protein abundance protein abundance
  • translational efficiency See e.g. Zhao et al. AJHG 2016, 98: 299-309.
  • Editing of coding sequences can be made using the methods disclosed herein to increase the level of preselected amino acids in the encoded polypeptide.
  • the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference.
  • Other proteins include methionine- rich plant proteins such as from sunflower seed (Lilley et al. Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed.
  • Applewhite American Oil Chemists Society, Champaign, Ill. 1989, pp. 497-502; herein incorporated by reference
  • corn Pedersen et al. J. Biol. Chem. 1986, 261: 6279; Kirihara et al. Gene 1988, 71: 359; both of which are herein incorporated by reference
  • rice agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.
  • the methods disclosed herein can be used to modify herbicide resistance traits including genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing DNA sequence modifications leading to such resistance, in particular the S4 and/or Hra modifications), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene); glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No.
  • ALS acetolactate synthase
  • ALS sulfonylurea-type herbicides
  • glutamine synthase such as phosphinothricin or basta
  • glyphosate e.g., the EPSPS
  • the bar gene encodes resistance to the herbicide basta
  • the nptll gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS-gene mutants encode resistance to the herbicide chlor sulfuron.
  • Sterility genes can also be modified and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development. Additional sterility traits are described for example in U.S. Patent Application 2016/0208243, herein incorporated by reference.
  • Genome editing can also be used to make haploid inducer lines as disclosed in WO20 18086623 and US20190292553.
  • the quality of grain can be altered by modifying genes encoding traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose.
  • modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
  • PHAs polyhyroxyalkanoates
  • Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like.
  • the level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
  • the methods disclosed herein can also be used for modification of native plant gene expression to achieve desirable plant traits, such as an agronomically desirable trait.
  • desirable plant traits include, for example, disease resistance, herbicide tolerance, drought tolerance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content.
  • edits generated by the methods provided herein are evaluated for changes in phenotype on a sub-organismal level, including evaluation of RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, and/or translational efficiency.
  • edits generated by the methods provided herein are evaluated for changes in phenotype on an organismal level, including yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, disease resistance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content.
  • the optimal allele and/or edits may be selected based on sub- organismal phenotype and/or organismal phenotype.
  • the present disclosure may be used for genomic editing of any plant species, including, but not limited to, monocots and dicots (i.e., monocotyledons and dicotyledons, respectively).
  • plant species of interest include, but are not limited to, com (Zea mays'), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale).
  • sorghum (Sorghum bicolor, Sorghum vulgare), camelina (Camelina sativa), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panic urn miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), quinoa (Chenopodium quinoa), chicory (Cichorium intybus), lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot
  • the graft is a heterograft. In other embodiments, the graft is a homograft. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and/or rootstock is a dicot. In some embodiments, the scion and/or rootstock is a monocot. In some embodiments, the scion is soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, or wheat.
  • the meristem is edited.
  • the genome of a meristem of a plant scion grafted onto a rootstock is edited.
  • At least one first target sequence is within an intergenic region and at least one second target sequence is within a genic region. In some embodiments, at least one first target sequence is within a genic region and at least one second target sequence is within an intergenic region. In some embodiments, at least one first target sequence is within an intergenic region and at least one second target sequence is within an intergenic region. In some embodiments, at least one first target sequence is within a genic region and at least one second target sequence is within a genic region. In some embodiments, at least one first target sequence is the same as at least one second target sequence. In some embodiments, at least one first target sequence is different from at least one second target sequence. In some embodiments, at least one first target sequence has at least 80%, at least
  • a genomic locus comprising at least one first target sequence is homologous to at least about 1, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, at least about 300 bp, at least about 350 bp, at least about 400 bp, at least about 450 bp, at least about 500 bp, at least about 600 bp, at least about 700 bp, at least about 800 bp, at least about 900 bp, or at least about 1000 bp of a genomic locus comprising at least one second target sequence.
  • a genomic locus comprising at least one first target sequence is homologous to at least about 1, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least
  • the target sequences are selected from genomic regions, between which a recombination event would exchange genetic materials between genomic regions provided by the male and female parents of the scion.
  • the reagents and methods described provide a relatively easy and convenient solution for producing plants with altered genomes, i.e., individuals with recombined DNA sequences designed in a targeted fashion.
  • the methods provided herein can be applied to edit one or more genomic regions selected independently from the group consisting of a gene, an array of tandemly duplicated genes, a multigene family, an enhancer, a suppressor, a promoter, a termination sequence, a splice acceptor sequence, a splice donor sequence, an intron, an exon, an siRNA, a sequence encoding a non-coding RNA, a microRNA, a transgene, and a quantitative trait locus (QTL).
  • QTL quantitative trait locus
  • recombination occurs by targeted DNA cleavage followed by homologous recombination.
  • DNA cleavage may be cleavage of one or both strands.
  • targeted DNA cleavage occurs at one or more target sequences (also referred to synonymously as “target loci”).
  • the edit results in the generation of a single stranded DNA break (SSB).
  • the edit results in the generation of a double stranded DNA break (DSB).
  • the edit results in recombination between target sequences in the scion genome.
  • recombination occurs between two target sequences.
  • the target sequences are on the same locus on homologous chromosomes. In some embodiments, the target sequences are on different loci on the same chromosome. In some embodiments, the target sequences are on different loci on homologous chromosomes. Embodiments in which the target sequences are in different loci may include simultaneously inducing independent targeted recombination events. [0212] In some embodiments, the method does not include providing a template for homology-directed repair (HDR).
  • HDR homology-directed repair
  • the edit results in new combinations of genomic sequences into the same chromosome. In some embodiments, the edit results in recombination at or near the target sequence. In some embodiments, the edit results in recombination at a site within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 nucleotides from the target sequence.
  • the edit results in recombination at a site within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12500, 15000, 17500, 20000, 22500, or 25000 nucleotides from the target sequence.
  • the edit results in recombination at or near the target sequence.
  • the methods and systems rely on DNA or RNA molecules produced with established molecular biology techniques.
  • the DNA or RNA molecules which comprise genome-editing reagents, are then introduced into a plant and taken up into meristematic cells.
  • the meristematic cell genomes are thus altered, and the DNA sequence modifications (e.g., targeted recombinations) are carried into germline cells and subsequent generations.
  • mutated seeds from plants edited with the reagents and methods described here are collected for phenotypic characterization.
  • pollen from edited plants is used in crosses with other individuals, or mutated individuals are pollinated with pollen of unedited plants or wildtype plants.
  • the embodiments described methods and reagents can have many advantages over other known solutions.
  • the techniques presented generally bypass callus induction or tissue culture that are necessary for alternative or widely practiced genome editing procedures, thus speeding up (i.e., accelerating) and lowering or reducing the cost of the process of producing plants with targeted DNA sequence modifications.
  • Epigenetic resetting i.e., interference
  • the editing can be performed in individuals of an elite genetic background, making lengthy backcrossing schemes unnecessary.
  • RNA molecules that comprise a sequence specific endonuclease, binding protein, or recombinase and/or guide nucleic acid(s) that are operably linked to MTS sequences are also provided herein.
  • guide nucleic acids are RNA molecules that will be present at detectable concentrations in the plants for only a certain period of time following a stimulus.
  • the concentrations of RNA molecules comprising guide RNAs separated by processing elements comprising direct repeats DR, i.e., pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA)
  • concentrations of RNA molecules comprising guide RNAs separated by processing elements comprising direct repeats which are capable of being processed by an RNA-guided nuclease are also expected to be decreased in tissues where the RNA-guided nuclease is located.
  • RNA molecules can be detected by a variety of techniques that include reverse transcription polymerase chain reaction (RT-PCR) assays where oligonucleotide primers and optionally detection probes which specifically amplify and detect the unprocessed RNA molecule comprising the Cas nuclease and/or guide RNA(s) that are operably linked to MTS sequences are used.
  • RT-PCR reverse transcription polymerase chain reaction
  • Such plants can comprise any of the RNA molecules or combinations of RNA molecules present in the compositions provided herein that are used to contact the plants.
  • an active form of the RNA guided nuclease is predominantly localized in meristem tissue of the plant.
  • the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises an operably linked MTS sequence. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is operably linked to promoters that include a rootpreferred or root-specific promoter which is active in root cells. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is operably linked to constitutively active promoters.
  • DNA encoding the RNA-guided nuclease can be provided in a transgene that is stably integrated in the genome of the plant, in DNA that is not integrated into the plant genome, or in DNA provided in a viral vector (e.g., a geminivirus replicon).
  • Geminivirus DNA replicons suitable for delivery of DNA molecules encoding an RNA-guided nuclease to plants include a Beet Yellow Dwarf Virus replicon (Baltes et al. Plant Cell 2014, 26(1): 151- 63; doi: 10.1105/tpc.113.119792).
  • an MTS is operably linked to a sequence specific endonuclease, binding protein, recombinase, and/or a guide nucleic acid(s) comprising a plurality of guide RNAs (e.g., 2, 3, 4, or more guide RNAs) separated by processing elements to provide for gene editing at a plurality of genomic locations targeted by each guide RNA.
  • a guide RNAs e.g., 2, 3, 4, or more guide RNAs
  • the plurality of guide RNAs are separated by processing elements comprising direct repeats (DR; i.e., pre-crRNAs comprising a full-length direct repeat (full- DR-crRNA)) which are capable of being processed (i.e., cleaved) by an RNA-guided nuclease.
  • DR direct repeats
  • pre-crRNAs comprising a full-length direct repeat (full- DR-crRNA)
  • full- DR-crRNA full-length direct repeat
  • DRs include the Casl2a DR (e.g., SEQ ID NO: 54 or 56) which can be cleaved by a Casl2a guided nuclease (e.g., SEQ ID NO: 53 or 55, respectively).
  • RNAs comprising Casl2a DRs by Casl2a have been described (Fonfara et al. Nature 2016, 532: 517-521, doi.org/10.1038/naturel7945); US20160208243; WO 2017/189308).
  • Other examples of such DRs include the Casl2j DRs (e.g., SEQ ID NO: 58,
  • a Casl2j guided nuclease (e.g., SEQ ID NO: 57, 59, or
  • the crRNA portion of the DR can remain as a part of the gRNA after processing and can be recognized by the RNA guided nuclease to provide for editing of genomic DNA recognized via hybridization of the gRNA to the targeted genomic site.
  • the meristem is part of a plant scion grafted onto a rootstock. In other embodiments, the meristem is part of a non-grafted plant.
  • the targeted recombination event in the scion is passed to a progeny in a seed produced by the scion.
  • the method further comprises subjecting one or more seeds and/or progeny comprising one or more targeted recombination events to one or more additional rounds of targeted recombination and/or selection.
  • the methods described herein facilitate targeted recombination.
  • the targeted recombination is between chromosomes of a heterozygous genome.
  • the targeted recombination is between chromosomes of a homozygous genome.
  • the method allows for more rapid generation of allelic diversity within a unit of inheritance than traditional breeding alone, such as, for example within a single generation.
  • the method allows for more rapid generation of one or more fixed alleles among a breeding population than traditional breeding alone.
  • the targeted recombination results in increased predicted genetic gains compared to predictions based on the same genome absent the targeted recombination.
  • genetic gain is measured as the expected increase in performance of a given trait through a selection and/or targeted recombination process, such as, e.g., as described in Xu Y, et al (2019). Enhancing Genetic Gain through Genomic Selection: From Livestock to Plants. Plant Commun. Oct 16; 1( 1): 100005.
  • genetic gain is measured as increased genetic diversity compared to a pool of potential parent plants or compared to the starting genome of the scion or the genomes of the parents of the scion.
  • Various methods and models for measuring or otherwise assessing genetic gain may be used in the claimed methods, including some that are crop- and/or traitdependent, such as, e.g., those laid out in Brandariz, S.P. and Bernardo, R. (2019), Predicted Genetic Gains from Targeted Recombination in Elite Biparental Maize Populations.
  • Vectors are used to deliver nucleic acids to plant cells.
  • the vector is capable of autonomous replication within the host cell.
  • the vector is integrated into the genome of the host cell and replicated with the host genome.
  • expression vectors termed “expression vectors”, the genes of the vector are expressed or are capable of being expressed under certain conditions.
  • the vector contains one or more regulatory elements operably linked to a gene.
  • the vector contains a promoter.
  • the promoter is a constitutive promoter, a conditional promoter, an inducible promoter, or a temporally or spatially specific promoter (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).
  • a vector is introduced to a host cell to produce RNA transcripts, proteins, or peptides within the host cell, as encoded by the contained nucleic acid.
  • the nucleic acid described herein can be contained within any suitable plant transformation plasmid or vector.
  • the plant transformation plasmid or vector further comprises a selectable or screenable marker, such as but not limited to a fluorescent protein.
  • the engineered system or a component thereof is delivered via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, geminiviruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus.
  • adenoviruses lentiviruses
  • adeno-associated viruses retroviruses
  • retroviruses geminiviruses
  • begomoviruses tobamoviruses
  • potex viruses comoviruses
  • wheat streak mosaic virus barley stripe mosaic virus
  • bean yellow dwarf virus bean pod mottle virus
  • cabbage leaf curl virus cabbage leaf curl virus
  • beet curly top virus tobacco yellow dwarf virus
  • the engineered system or a component thereof is delivered via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp.
  • a viral vector may be delivered to a plant by transformation with Agrobacterium.
  • a T-DNA vector is used to deliver at least one nucleic acid to plant cells.
  • a T-DNA binary vector is used.
  • a T-DNA superbinary vector system is used.
  • a T-DNA ternary vector system is used.
  • the T-DNA system further comprises an additional virulence gene cluster.
  • the T-DNA system further comprises an accessory plasmid or virulence helper plasmid.
  • the T-DNA vector is an Agrobacterium vector.
  • the T-DNA vector is an Agrobacterium rhizogenes vector.
  • Agrobacterium rhizogenes also known as Rhizobium rhizogenes, is a gram-negative soil bacteria that is capable of infecting the roots of a variety of plant species. Transformation of cells of the plant root with the Ri (root inducing) plasmid of the bacteria results in random integration of the genes from the Ri plasmid into the plant cell genome. This leads to expression of the genes from the Ri plasmid in the cells of the root, resulting in the host plant producing branching root overgrowth at the site of infection in what is known as “hairy root syndrome”. Replacement of the genes of the Ri plasmid with the desired transformation product, while maintaining the virulence genes, results in the ability to produce transgenic roots that are express the genes of the desired transformation product.
  • the nucleic acid encoding the Cas nuclease and the nucleic acid encoding the guide RNA are provided in the same vector. In some embodiments, the nucleic acid encoding the Cas nuclease and the nucleic acid encoding the guide RNA are provided in different vectors. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral vector or a T-DNA vector.
  • the plant cell is a cell of a rootstock.
  • the plant cell is a cell of a grafted scion.
  • the plant cell is a cell of a seed (including mature seed and immature seed).
  • the plant cell is a cell of a plant cutting.
  • the plant cell is a cell of a plant cell culture.
  • the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers,
  • one or more treatments is employed to deliver genome editing reagents into a plant cell or plant protoplast, e.g., through barriers such as a cell wall or a plasma membrane or nuclear envelope or other lipid bilayer.
  • genome editing reagents are delivered directly, for example by direct contact of the polynucleotide composition with a plant cell or plant protoplast.
  • a genome editing reagent-containing composition in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant cell or plant protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection).
  • a plant cell or plant protoplast is soaked in a liquid genome editing reagent-containing composition, whereby the genome editing reagent is delivered to the plant cell or plant protoplast.
  • the genome editing reagent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure.
  • the genome editing reagent-containing composition is introduced into a plant cell or plant protoplast e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in U.S. Published Patent Application 2014/0287509, incorporated by reference in its entirety herein.
  • Other techniques useful for delivering the genome editing reagent-containing composition to a plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation.
  • ultrasound or sonication vibration, friction, shear stress, vortexing, cavitation
  • centrifugation or application of mechanical force e.g., mechanical cell wall or cell membrane deformation or breakage
  • enzymatic cell wall or cell membrane breakage or permeabilization e.g., abrasion with carborundum or other particulate abrasive or scar
  • the genome editing reagent-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the gRNA; see, e.g., Broothaerts et al. Nature 2005, 433: 629-633.
  • bacterially mediated e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.
  • any of these techniques or a combination thereof are alternatively employed on the plant part or tissue or intact plant (or seed) from which a plant cell or plant protoplast is optionally subsequently obtained or isolated; in embodiments, the genome editing reagent-containing composition is delivered in a separate step after the plant cell or plant protoplast has been obtained or isolated.
  • a treatment employed in delivery of a genome editing reagent to a plant cell or plant protoplast is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the plant cell or plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the plant cell or plant protoplast occurs), or treating at a combination of different temperatures.
  • a specific thermal regime is carried out on a plant cell or plant protoplast, or on a plant or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the genome editing reagent delivery.
  • a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cutting. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cell culture.
  • a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon,
  • a whole plant or plant part or seed, or an isolated plant cell or plant protoplast, or the plant or plant part from which a plant cell or plant protoplast is obtained or isolated is treated with one or more delivery agents which can include at least one chemical, enzymatic, or physical agent, or a combination thereof.
  • a genome editing reagent-containing composition further includes one or more one chemical, enzymatic, or physical agent for delivery.
  • the treated plant cell is a cell of a rootstock.
  • the treated plant cell is a cell of a grafted scion.
  • the treated plant cell is a cell of a seed (including mature seed and immature seed).
  • the treated plant cell is a cell of a plant cutting. In some embodiments, the treated plant cell is a cell of a plant cell culture. In some embodiments, the treated plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovul
  • Treatment with the chemical, enzymatic or physical agent can be carried out simultaneously with the genome editing reagent delivery, or in one or more separate steps that precede or follow the genome editing reagent delivery.
  • a chemical, enzymatic, or physical agent, or a combination of these is associated or complexed with a genome editing reagent composition; examples of such associations or complexes include those involving non-covalent interactions (e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles, or other heterogeneous composition) and covalent interactions (e.g., peptide bonds, bonds formed using cross-linking agents).
  • a genome editing reagent is provided as a liposomal complex with a cationic lipid, or as a complex with a carbon nanotube, or as a fusion protein between the nuclease and a cell-penetrating peptide.
  • agents useful for delivering a genome editing reagent include the various cationic liposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J Controlled Release, 123:1-10, and the cross-linked multilamellar liposomes described in U.S. Patent Application Publication 2014/0356414 Al, incorporated by reference in its entirety herein.
  • compositions comprising: (i) RNA molecules comprising an MTS operably linked to, e.g., a sequence specific endonuclease, binding protein, or recombinase and/or guide nucleic acid(s), such as, for instance, a Cas nuclease and/or guide RNA(s) ; (ii) nucleic acids encoding sequence specific endonucleases, binding proteins, and/or recombinase and/or guide nucleic acid(s); and/or (iii) donor DNA templates can further comprise components that include:
  • solvents e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems
  • solvents e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems
  • fluorocarbons e.g., perfluorodecalin, perfluoromethyldecalin
  • glycols or polyols e.g., propylene glycol, polyethylene glycol
  • surfactants including cationic surfactants, anionic surfactants, non-ionic surfactants, and amphiphilic surfactants, e.g., alkyl or aryl sulfates, phosphates, sulfonates, or carboxylates; primary, secondary, or tertiary amines; quaternary ammonium salts; sultaines, betaines; cationic lipids; phospholipids; tallowamine; bile acids such as cholic acid; saponins or glycosylated triterpenoids or glycosylated sterols (e.g., saponin commercially available as catalogue number 47036-50g-F, Sigma-Aldrich, St.
  • surfactants including cationic surfactants, anionic surfactants, non-ionic surfactants, and amphiphilic surfactants, e.g., alkyl or aryl sulfates, phosphates, sulfon
  • organosilicone surfactants including nonionic organosilicone surfactants such as trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as SIL WET L-77TM brand surfactant having CAS Number 27306- 78-1 and EPA Number CAL. REG. NO.
  • surfactants include sodium lauryl sulfate, the Tween series of surfactants, Triton-XlOO, Triton-X114, CHAPS and CHAPSO, Tergitol-type NP-40, and Nonidet P-40;
  • peptides, proteins, or enzymes e.g., cellulase, pectolyase, maceroenzyme, pectinase
  • cell-penetrating or pore-forming peptides e. g., (B0100)2K8, Genscript; polylysine, poly-arginine, or poly -homoarginine peptides; gamma zein, see US Patent Application publication 2011/0247100, incorporated herein by reference in its entirety; transcription activator of human immunodeficiency virus type 1 (“HIV-1 Tat”) and other Tat proteins, see, e.
  • HIV-1 Tat human immunodeficiency virus type 1
  • cationic branched or linear polymers such as chitosan, poly-lysine, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”, e. g., PEI, branched, MW 25,000, CAS# 9002-98-6; PEI, linear, MW 5000, CAS# 9002-98-6; PEI linear, MW 2500, CAS# 9002-98-6);
  • (k) counter-ions amines or polyamines (e. g., spermine, spermidine, putrescine), osmolytes, buffers, and salts (e. g., calcium phosphate, ammonium phosphate);
  • polynucleotides e. g., non-specific double- stranded DNA, salmon sperm DNA
  • transfection agents e. g., Lipofectin®, Lipofectamine®, and Oligofectamine®, and Invivofectamine® (all from Thermo Fisher Scientific, Waltham, MA), PepFect (see Ezzat et al. Nucleic Acids Res. 2011, 39: 5284 - 5298), Transit® transfection reagents (Minis Bio, LLC, Madison, WI), and poly-lysine, poly-homoarginine, and poly-arginine molecules including octo-arginine and nono-arginine as described in Lu et al. J. Agric. Food Chem. 2010, 58: 2288 - 2294);
  • transfection agents e. g., Lipofectin®, Lipofectamine®, and Oligofectamine®, and Invivofectamine® (all from Thermo Fisher Scientific, Waltham, MA), PepFect (see Ezzat et al. Nucleic Acids Res. 2011, 39: 5284 - 52
  • antibiotics including non-specific DNA double- strand-break- inducing agents (e. g., phleomycin, bleomycin, talisomycin);
  • antioxidants e. g., glutathione, dithiothreitol, ascorbate
  • chelating agents e. g., EDTA, EGTA.
  • the chemical agent is provided simultaneously with the genome editing reagent.
  • the genome editing reagent is covalently or non-covalently linked or complexed with one or more chemical agent; for example, a polynucleotide genome editing reagent can be covalently linked to a peptide or protein (e.g., a cell-penetrating peptide or a pore-forming peptide) or non-covalently complexed with cationic lipids, polycations (e.g., polyamines), or cationic polymers (e.g., PEI).
  • the genome editing reagent is complexed with one or more chemical agents to form, e.g., a solution, liposome, micelle, emulsion, reverse emulsion, suspension, colloid, or gel.
  • the physical agent is at least one selected from the group consisting of particles or nanoparticles (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics) in various size ranges and shapes, magnetic particles or nanoparticles (e.g., silenceMag MagnetotransfectionTM agent, OZ Biosciences, San Diego, Calif.), abrasive or scarifying agents, needles or microneedles, matrices, and grids.
  • particulates and nanoparticulates are useful in delivery of the polynucleotide composition or the nuclease or both.
  • Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomplexes, or nanotubes), and composites (e.g., polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene, palladium/graphene nanocomposites).
  • metals e.g., gold, silver, tungsten, iron, cerium
  • ceramics e.g., aluminum oxide, silicon carbide, silicon
  • such particulates and nanoparticulates are further covalently or non-covalently functionalized, or further include modifiers or cross-linked materials such as polymers (e.g., linear or branched polyethylenimine, poly-lysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, polyglycols (e.g., polyethylene glycol, thiolated polyethylene glycol), polypeptides or proteins, and detectable labels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot).
  • polymers e.g., linear or branched polyethylenimine, poly-lysine
  • polynucleotides e.g., DNA or RNA
  • polysaccharides e.g., DNA or RNA
  • lipids lipids
  • polyglycols e.g., polyethylene glycol, thiolated polyethylene glycol
  • Embodiments of compositions including particulates include those formulated, e.g., as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids.
  • Embodiments include nanoparticles affixed to a surface or support, e.g., an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate.
  • Embodiments include polynucleotide compositions including particulates (e.g., gold or tungsten or magnetic particles) delivered by a Biolistic-type technique or with magnetic force.
  • the size of the particles used in Biolistics is generally in the “microparticle” range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micrometer size ranges (see, e.g., instruction manual for the Helios® Gene Gun System, Bio-Rad, Hercules, Calif.; Randolph- Anderson et al. (2015) “Sub-micron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types”, Bio-Rad US/EG Bulletin 2015), but successful Biolistics delivery using larger (40 nanometer) nanoparticles has been reported in cultured animal cells; see O'Brian and Lummis (2011) BMC Biotechnol., 11:66- 71.
  • nanoparticles which are generally in the nanometer (nm) size range or less than 1 micrometer, e.g., with a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm.
  • nanoparticles commercially available (all from Sigma- Aldrich Corp., St.
  • Louis, Mo. include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” of less than 25 nm particle size; single-, double-, and multi-walled carbon nanotubes, e.g., with diameters of 0.7-1.1, 1.3-2.3, 0.7-0.9, or 0.7-1.3 nm, or with nanotube bundle dimensions of 2-10 nm by 1-5 micrometers, 6-9 nm by 5 micrometers, 7-15 nm by 0.5-10 micrometers, 7-12 nm by 0.5-10 micrometers, 110-170 nm by 5-9 micrometers, 6-13 nm by 2.5-20 micrometers.
  • Embodiments include genome editing reagent-containing compositions including materials such as gold, silicon, cerium, or carbon, e.g., gold or gold- coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g., carbon nanotubes functionalized with amide, amino, carboxylic acid, sulfonic acid, or polyethylene glycol moieties), and graphene or graphene oxide or graphene complexes; see, for example, Wong et al.
  • materials such as gold, silicon, cerium, or carbon, e.g., gold or gold- coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles,
  • a genome editing reagent is delivered to plant cells or plant protoplasts prepared or obtained from a plant, plant part, or plant tissue that has been treated with the polynucleotide compositions (and optionally the nuclease).
  • the treated plant cell is a cell of a rootstock.
  • the treated plant cell is a cell of a grafted scion.
  • the treated plant cell is a cell of a seed (including mature seed and immature seed).
  • the treated plant cell is a cell of a plant cutting.
  • the treated plant cell is a cell of a plant cell culture.
  • the treated plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers
  • one or more one chemical, enzymatic, or physical agent, separately or in combination with the genome editing reagent is provided/applied at a location in the plant or plant part other than the plant location, part, or tissue from which the plant cell or plant protoplast is obtained or isolated.
  • the genome editing reagent is applied to adjacent or distal cells or tissues and is transported (e.g., through the
  • a genome editing reagent-containing composition is applied by soaking a seed or seed fragment or zygotic or somatic embryo in the genome editing reagent-containing composition, whereby the genome editing reagent is delivered to the seed or seed fragment or zygotic or somatic embryo from which plant cells or plant protoplasts are subsequently isolated.
  • a flower bud or shoot tip is contacted with a genome editing reagent-containing composition, whereby the genome editing reagent is delivered to cells in the flower bud or shoot tip from which plant cells or plant protoplasts are subsequently isolated.
  • a genome editing reagentcontaining composition is applied to the surface of a plant or of a part of a plant (e.g., a leaf surface), whereby the genome editing reagent is delivered to tissues of the plant from which plant cells or plant protoplasts are subsequently isolated.
  • a whole plant or plant tissue is subjected to particle- or nanoparticle-mediated delivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery) of a genome editing reagent-containing composition, whereby the genome editing reagent is delivered to cells or tissues from which plant cells or plant protoplasts are subsequently isolated.
  • compositions comprising: (i) RNA molecules comprising an MTS operably linked to a sequence specific endonuclease, binding protein, or recombinase, and/or a guide nucleic acid(s); (ii) nucleic acids encoding sequence specific endonucleases, binding proteins, or recombinase and/or guide nucleic acid(s); and/or (iii) donor DNA templates can be delivered to the plant and/or meristem cells of the plant by particle mediated delivery, and any other direct method of delivery, such as but not limiting to, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, and/or by use of cellpenetrating peptides.
  • Agrobacterium-mediated transformation polyethylene glycol (PEG)-mediated transfection to protoplasts
  • whiskers mediated transformation electroporation, particle bombardment, and/or by use of cellpen
  • the plant cell to which the composition is delivered is a cell of a rootstock. In some embodiments, the plant cell to which the composition is delivered is a cell of a grafted scion. In some embodiments, the plant cell to which the composition is delivered is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell to which the composition is delivered is a cell of a plant cutting. In some embodiments, the plant cell to which the composition is delivered is a cell of a plant cell culture.
  • the plant cell to which the composition is delivered is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tub
  • plants are contacted either simultaneously or sequentially with one, two, three or more RNA molecules in one or more compositions where at least one of the RNA molecules comprises a guide nucleic acid fused to an MTS.
  • the composition contacts a rootstock.
  • the composition contacts a grafted scion.
  • the composition contacts a seed (including mature seed and immature seed).
  • the composition contacts a plant cutting.
  • the composition contacts a plant cell culture.
  • the composition contacts a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots,
  • plants are contacted either simultaneously or sequentially with one, two, three or more nucleic acid molecules in one or more compositions where at least one of the nucleic acid molecules comprises an RNA encoding a sequence specific endonuclease, binding protein, or recombinase fused to an MTS.
  • one of the guide nucleic acid molecules comprises a guide RNA fused to an MTS and a second RNA molecule comprises RNA encoding an RNA guided endonuclease and optionally an MTS, where the RNA guided endonuclease can process the RNA comprising the guide RNA to release a functional guide RNA.
  • one of the RNA molecules comprises at least one guide nucleic acid fused to an MTS and a second guide nucleic acid molecule comprises RNA encoding an RNA guided nuclease and optionally an MTS, where the RNA guided nuclease cannot process the RNA comprising the guide RNA to release a functional guide RNA (e.g., processing elements present in the RNA molecule comprising the gRNA and the MTS are not recognized by the RNA-guided nuclease).
  • a functional guide RNA e.g., processing elements present in the RNA molecule comprising the gRNA and the MTS are not recognized by the RNA-guided nuclease.
  • guide RNAs of the first and second RNA molecule are flanked by or comprise processing elements (e.g., DRs) which are processed by different RNA-guided nuclease (e.g., a Casl2a nuclease can process the first RNA molecule and a Casl2j nuclease can process the second RNA molecule).
  • processing elements e.g., DRs
  • different RNA-guided nuclease e.g., a Casl2a nuclease can process the first RNA molecule and a Casl2j nuclease can process the second RNA molecule.
  • the guide RNA(s) of the first RNA molecule distinct from the guide RNA(s) of the second RNA molecule.
  • Such distinct gRNAs provided by the first RNA molecule can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second RNA molecule can provide for genome editing at one or more second genomic sites in a meristem cell.
  • Such contacting the plant with RNA molecules in a composition can occur sequentially such that the first gRNA(s) are delivered, allowed sufficient time (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours) to effect desired genome edits, followed by contacting the plant with the second RNA molecules in a second composition to deliver the second gRNA(s) to effect additional desired genome edits, where such desired genome edits are effected by providing the gRNA(s) and an RNA guided nuclease in at least the meristem cell.
  • a plant can be contacted by one or more RNA molecules that comprise at least one gRNA fused to an MTS, optionally along with an RNA encoding RNA guided Cas nuclease, permitted a sufficient period of time to accumulate the RNA molecule in the meristem cells (e.g., about 6, 12, 18 or 20 to about 24, 30, or 36 hours apart), and then contacted with a different mixture of one or more RNA molecules that comprise at least one different gRNA fused to an MTS, optionally along with an RNA encoding an RNA guided Cas nuclease, where the RNA guided Cas nuclease can process the RNA comprising the guide RNA to release a functional guide RNA and/or effect a desired genomic edit with the gRNA in the meristem cells.
  • Guide nucleic acids can be provided to at least the meristem cell by a variety of methods that include stable expression with an integrated transgene, expression from a viral vector, or transient expression such as by introducing an RNA that encodes the gRNA or a DNA that encodes the gRNA that is operably linked to an MTS.
  • the gRNA is predominantly localized in meristem tissue of the plant.
  • RNAs encoding the gRNA(s) or DNA(s) that encode those gRNA(s) to the plant and/or meristem cells of the plant can be achieved by particle mediated delivery, and any other direct method of delivery, such as but not limited to, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, and/or by use of cell-penetrating peptides.
  • the guide nucleic acid(s) are delivered to a rootstock. In some embodiments, the guide nucleic acid(s) are delivered to a grafted scion.
  • the guide nucleic acid(s) are delivered to a seed (including mature seed and immature seed). In some embodiments, the gRNA(s) are delivered to a plant cutting. In some embodiments, the guide nucleic acid(s) are delivered to a plant cell culture.
  • the guide nucleic acid(s) are delivered to a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).
  • a plant organ e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber,
  • a guide nucleic acid for a sequence specific endonuclease, binding protein, or recombinase is applied to a leaf, a shoot, a stem, and/or meristem of the plant.
  • a composition comprising guide nucleic acid(s) for the sequence specific endonuclease, binding protein, or recombinase is applied to a leaf, a shoot, a stem, and/or meristem of the plant.
  • the composition comprising the guide nucleic acid(s) comprises a nuclease inhibitor.
  • the composition comprising the guide nucleic acid(s) comprises an RNase inhibitor.
  • delivery of the guide nucleic acid(s) comprises spraying a composition comprising the guide nucleic acid(s) onto the leaves, shoot, stem, and/or meristem.
  • the composition comprising the guide nucleic acid(s) comprises a surfactant.
  • the composition comprising the guide nucleic acid(s) comprises glass beads coated with the guide nucleic acid(s).
  • delivery of the guide nucleic acid(s) comprises rubbing a composition comprising the guide nucleic acid(s) onto the leaves, shoot, stem, and/or meristem.
  • delivery of the guide nucleic acid(s) comprises injecting a composition comprising the guide nucleic acid(s) into the stem.
  • delivery of the guide nucleic acid(s) comprises leaf infiltration of a composition comprising the guide nucleic acid(s) into a leaf.
  • the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • delivery of a guide nucleic acid(s) for the sequence specific endonuclease, binding protein, or recombinase nuclease comprises biolistic transformation of nucleic acid encoding the guide nucleic acid(s) into the leaf, shoot, stem, and/or meristem.
  • the biolistic transformation comprises transformation of circular DNA encoding the guide nucleic acid(s).
  • a guide nucleic acid(s) for the sequence specific endonuclease, binding protein, or recombinase is delivered to the roots of the plant.
  • a composition comprising the guide nucleic acid(s) for the sequence specific endonuclease, binding protein, or recombinase is applied to the roots.
  • the composition comprising the guide nucleic acid(s) comprises a nuclease inhibitor.
  • the composition comprising the guide nucleic acid(s) comprises an RNase inhibitor.
  • the guide nucleic acid(s) is delivered to the plant root by incubating the root with a composition comprising the guide nucleic acid(s).
  • a guide nucleic acid(s) for the sequence specific endonuclease, binding protein, or recombinase is delivered to the plant root by Agrobacterium rhizogenes transformation.
  • Sequence specific endonucleases, binding proteins, and/or recombinases can be provided to at least the meristem cell by a variety of methods that include stable expression with an integrated transgene, expression from a viral vector, or transient expression such as by introducing an RNA that encodes the sequence specific endonuclease, binding protein, and/or recombinase or an RNA that encodes the sequence specific endonuclease, binding protein, and/or recombinase that is operably linked to an MTS.
  • an active form of the sequence specific endonuclease, binding protein, and/or recombinase is predominantly localized in root tissue of the plant.
  • sequence specific endonuclease, binding protein, and/or recombinase can be operably linked to a vegetative stage, root-preferred or root-specific promoter including but not limited to those disclosed in US Patent No. 8,058,419; US Patent No. 10,533,184; Khandal et al. Plant Biotechnol J 2020, 18: 2225-2240; Xu et al. Plant Biotechnol J 2020, 18: 1585-1597; and James et al. Front Plant Sci 2022, 13: 1009487.
  • a plant expressing transgenically a sequence specific endonuclease, binding protein, and/or recombinase may be genomically edited by delivery of a second nucleic containing only guide RNAs or guide DNAs suitable for the transgenically expressed sequence specific endonuclease, binding protein, and/or recombinase.
  • RNA sequences are generally made and assembled at first in DNA form as RNA expressing vectors using recombinant DNA technology. RNA expression is performed in vitro, and the RNA purified according to well established methods. Addition of 5’ caps and polyA tails to mRNAs can be performed according to methods established in the literature. Alternatively, some RNAs designed as described can be purchased from commercial providers.
  • a substantially purified RNA composition is understood to comprise a high concentration of an RNA molecule of interest, although in some cases it may comprise two distinct RNAs.
  • one RNA may comprise a sequence specific endonuclease, binding protein, or recombinase while another may comprise a corresponding guide or guide array.
  • a substantially purified RNA composition may comprise other added components, such as a pH buffer, salt, surfactants, and/or RNase inhibitors.
  • Plants can be effectively contacted with the RNA vectors in many ways. Often it will be convenient to load them into the phloem of plants through the leaves, for example by nicking a leaf and submerging the injured tissue into a solution of substantially purified RNAs. Other avenues are also possible, such as by injection into the stems with a needle or use of a handheld biolistics device. In some embodiments, a surfactant is added to the purified RNA, and the liquid is applied to a tissue like embryonic shoot, leaf, stem, or inflorescence, with or without slight injury such as scratching.
  • RNAs are often applied at the vegetative stage of the life cycle of a plant, so as to reach vegetative meristems before they convert to floral meristems. In some cases, however, it may be convenient to apply the vectors, RNA molecules, or compositions comprising the RNA molecules or vectors, to floral meristems, especially at early stages of differentiation.
  • a soybean plant is contacted at the vegetative stage with a composition comprising the RNA molecules or vectors at vegetative stage Ve, VI, or V2 to about the V4 V(n) stage where 1, 2, 3, 4, or n is the number of trifoliate leaves (Soybean Growth and Development, M. Licht, 2014, Iowa State University Extension and Outreach, PM 1945).
  • a maize plant is contacted at the vegetative stage with a composition comprising the RNA molecules or vectors at vegetative stage Ve, VI, or V2 to about the V4 V(n) stage (Com Growth Stages, M. Licht, Iowa State University Extension and Outreach, on the https internet site “crops[dot]extension[dot]iastate[dot]edu/encyclopedia/corn-growth- stages”).
  • Provided herein are methods of producing plants that are homozygous for an allele comprising grafting a scion onto a rootstock that expresses a mobile sequence specific endonuclease, and inducing haplodiploidy in the edited scion or editing an already haploid scion.
  • methods of producing plants that are homozygous for a particular allele produced using targeted recombination comprising grafting a scion onto a rootstock that expresses a mobile Cas enzyme, providing guide RNA for the Cas enzyme, and inducing haplodiploidy in the edited scion or editing an already haploid scion.
  • methods of producing plants that are homozygous for a particular allele produced using targeted recombination comprising grafting a scion onto a rootstock that expresses a mobile sequence specific binding protein fused to a recombinase, and inducing haplodiploidy in the edited scion or editing an already haploid scion.
  • provided herein are methods of producing plants that are homozygous for a particular allele produced using targeted recombination comprising grafting a scion onto a rootstock that expresses a mobile inactive Cas enzyme fused to a recombinase, providing guide RNA for the inactive Cas enzyme, and inducing haplodiploidy in the edited scion or editing an already haploid scion.
  • Homozygous plants comprising a particular allele produced using targeted recombination can then be produced by increasing the ploidy of the edited haploid tissue. Combining the grafting and editing technology is then followed by inducing haploidy, followed by regeneration of a diploid that then is homozygous for the at least one particular allele produced using targeted recombination. This is accomplished in the same starting plant generation or one progeny generation, rather than many generations and rounds of plant breeding.
  • a method of producing a plant homozygous for a particular allele produced using targeted recombination is provided.
  • the method includes a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the particular allele produced using targeted recombination; and c) treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed or the plant produced from the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination.
  • MTS meristem transport segment
  • the method includes a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the particular allele produced using targeted recombination; and c) treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed or the plant produced from the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination.
  • MTS meristem transport
  • the method includes a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the particular allele produced using targeted recombination; and c) treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed or the plant produced from the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination.
  • MTS meristem transport segment
  • a method of producing a plant homozygous for a particular allele produced using targeted recombination including a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) growing a diploid progeny from the scion; c) crossing a plant grown from the diploid progeny with a haploid inducer line to produce a haploid seed, wherein the haploid seed includes particular allele produced using targeted recombination; and d) treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed
  • a method of producing a plant homozygous for a particular allele produced using targeted recombination including a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) growing a diploid progeny from the scion; c) crossing a plant grown from the diploid progeny with a haploid inducer line to produce a haploid seed, wherein the haploid seed includes the particular allele produced using targeted recombination; and d) treating the haploid seed or a plant produced from the haploid seed
  • a method of producing a plant homozygous for a particular allele produced using targeted recombination including a) grafting a scion onto a rootstock including nucleic acid encoding a sequence specific recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; b) growing a diploid progeny from the scion; c) crossing a plant grown from the diploid progeny with a haploid inducer line to produce a haploid seed, wherein the haploid seed includes the particular allele produced using targeted recombination; and d) treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid
  • haploid progeny occurs naturally in land plants but is an extremely rare event. Over the last half-century, multiple genes and quantitative trait loci have been identified in crop plants that interfere with typical genomic maintenance and produce progeny that lacks genetic material of a parent expressing the haploid induction gene. In some embodiments, to rapidly produce fixed traits in a plant, scions that are diploid are crossed with a haploid induction line.
  • the scion tissue includes a diploid plant or plant part thereof, and haploid induction is performed to produce first generation haploid progeny of the diploid plant or plant part thereof. Editing of the scion can occur prior to haploid induction, following haploid induction, or in the same step as haploid induction.
  • Methods of haploid induction are various and established in the art.
  • haploid induction is employed in diploid starting tissue, a common protocol includes a cross. This form of haploid induction is often directionally performed, wherein one of either the seed-bearing plant(s) or pollen-bearing plant(s) in the cross expresses at least one haploid induction gene.
  • the resultant progeny is haploid as a result of the haploid induction genes’ activity, and genetic material of the haploid inducer line does not contribute to the genetic content of the resultant progeny.
  • Maternal haploidy is induced by the pollinator parent, wherein the pollen is the haploid inducer line.
  • the pollen In inducing maternal haploidy, the pollen does not contribute genomic content to the progeny of the cross, and the produced haploid line is genetically based on the seed-bearing plant.
  • the haploid inducer line includes pollen.
  • the haploid inducer line does not contribute genomic content to the haploid seed.
  • the pollen comprises one or more modified genes that result in haploid formation when the pollen is used to fertilize an egg cell.
  • QTLs quantitative trait loci
  • qhirl Quantitative Haploid Induction Rate 1
  • qhir8 Quantitative Haploid Induction Rate 8
  • qhir2-qhir7 Genomic techniques established in the art enable the cloning of QTLs that contribute to increased haploid induction rates.
  • the haploid inducer line includes pollen with a com quantitative trait locus selected from the group consisting of qhir2, qhir3, qhir4, qhir5, qhir6, qhir7, qhir8, qmhirl , qmhir2, qhmfl , qhmf2, qhmf3, and qhmf4.
  • the haploid inducer comprises a trans gene or a mutation in a gene associated with chromosome segregation.
  • haploid-inducing gene such as qhirl being cloned as MATRILINEAL (ZmMTL), which is also called NOT LIKE DAD (NLD) or PHOSPHOPILASE A ⁇ (ZmPLA 1) and produces a pollen- specific phospholipase (Wang 2022).
  • ZmMTL MATRILINEAL
  • NLD NOT LIKE DAD
  • ZmPLA 1 PHOSPHOPILASE A ⁇
  • the haploid inducer line includes a mutated haploid inducing gene selected from the group consisting of MTL, ZMPLA1, NLD, INDETERMINATE GAMETOPHYTE (IG), HAPLOID INITIATION (HAP), CENTROMERE-SPECIFIC HISTONE 3 (CENH3), FIRST DIVISION RESTIUTION 1 (FDR1), OMISSION OF SECOND DIVISION 1 (OSD1), TOPOISOMERASE-LIKE ENZYME 1 (SPO11-1), or MEIOTIC RECOMBINATION GENE REC8 (REC8).
  • the haploid inducing gene is a transgene or a mutated gene.
  • Paternal haploidy is induced by the seed-bearing parent, where in the seed-bearing plant is the haploid inducer line.
  • the seed-bearing plant contributes cytoplasm but no genomic content to the progeny of the cross, and the produced haploid line is genetically based on the pollen-bearing plant.
  • the haploid inducer line includes a seed-bearing plant. In some embodiments, the haploid inducer line does not contribute genomic content to the haploid seed.
  • the haploid inducer line includes a mutated haploid inducing gene selected from the group consisting of MTL, ZMPLA1, NLD, IG, HAP, CENH3, FDR1, OSD1, SPO11-1, or REC8.
  • haploid induction through crossing in the present disclosure is accomplished through a cross with the edited scion. In other embodiments, haploid induction through crossing is accomplished through a cross with at least one progeny of the edited scion.
  • haploids Other methods of inducing haploidy are established in the art. Some of these methods include many techniques that can mutate specific genes, such as kinetochore or centromereregulating genes. Examples of such methods include ionizing radiation from x-rays or gamma radiation, non-ionizing radiation. In some embodiments, the scion is crossed with a non-haploid inducer line, and the method producing the haploid seed includes use of ionizing radiation. In some embodiments, the scion is pollinated with a non-haploid inducer line, and the method producing the haploid seed includes use of non-ionizing radiation.
  • haploid tissue involves selecting plant tissue from stages of the plant life cycle that are already haploid, such as pollen or egg cells. Traditional tissue regeneration methods can then be applied to tissue. Utilizing plant life cycle haploid stages of the life cycle involves the blockage of the normal development of these haploid cells, whose natural fate is the production of functional gametes or accessory cells, and their in vitro reprogramming towards a different developmental fate, which is to become embryos without fertilization (Segui-Simarro, Jacquier and Widiez (2021) Doubled Haploid Technology, 2287: 3-22).
  • Androgenesis is exemplified in this through culturing either pollen itself (producing a pollen culture) or wounding an anther then culturing it (producing a callus anther culture).
  • Gynogenesis is exemplified in this through culturing egg cells that have been treated with pressure or heat/cold shock during meiosis.
  • one method of producing a plant homozygous for a particular allele produced using targeted recombination includes grafting a haploid scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing an edited haploid cell in the scion, wherein the edited haploid cell comprises a targeted recombination event; culturing the edited cell in vitro to produce an edited haploid plant; and treating the edited haploid plant with an agent to increase ploidy in the edited haploid plant, thereby producing a plant homozygous for the gene edit.
  • MTS meristem transport segment
  • one method of producing a plant homozygous for a particular allele produced using targeted recombination includes grafting a haploid scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing an edited haploid cell in the scion, wherein the edited haploid cell comprises a targeted recombination event; culturing the edited cell in vitro to produce an edited haploid plant; and treating the edited haploid plant with an agent to increase ploidy in the edited haploid plant, thereby producing a plant homozygous for the gene edit.
  • MTS meristem transport segment
  • one method of producing a plant homozygous for a particular allele produced using targeted recombination includes grafting a haploid scion onto a rootstock including nucleic acid encoding a sequence specific recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing an edited haploid cell in the scion, wherein the edited haploid cell comprises a targeted recombination event; culturing the edited cell in vitro to produce an edited haploid plant; and treating the edited haploid plant with an agent to increase ploidy in the edited haploid plant, thereby producing a plant homozygous for the particular allele produced using targeted recombination.
  • plants homozygous for at least one allele produced using targeted recombination are produced within one plant generation.
  • an edited scion of the present disclosure is haploid
  • preparing the scion for chromosomal doubling in some embodiments involves in-vitro culturing and regeneration of the edited plant. Methods of doing so are established in the art and can be found in, for example, Segui-Simarro, Jacquier, and Widiez (2021) "Overview of in vitro and in vivo doubled haploid technologies.” Doubled. Haploid Technology: Volume 1: General Topics, Alliaceae, Cereals: 3-22. Vectors and in vitro culture methods for plant regeneration and regeneration of transformed plant tissue are available, for example, Gruber et al.
  • Regeneration can be accomplished through many different types of plant cells and plant tissues, such as plant callus, cultures, organs, pollen, embryos or parts thereof. Such regeneration techniques are generally described by Klee et al. (1987) Ann. Rev. of Plant Phys. 38: 467-486.
  • the scion is obtained through tissue culture. In some embodiments, the scion is obtained as a seedling. In some embodiments, the scion is grown from seed by tissue culturing. In some embodiments, the scion is grown from seed on germination paper. In some embodiments, the scion is grown in soil. In some embodiments, culturing the edited cell or edited cells in-vitro includes tissue culturing. For regeneration on tissue culture, the culture can contain certain plant hormones and energy sources or just energy sources. The growth medium may also contain a selection agent such as a biocide and/or an herbicide. Accordingly, some embodiments include selecting the treated seeds or plants including the particular allele produced using targeted recombination.
  • the nucleic acid encoding material for gene editing includes a reporter gene.
  • the reporter gene encodes a fluorescent protein.
  • the nucleic acid comprises a marker gene.
  • the marker gene encodes an antibiotic -resistant protein or an herbicide-resistant protein. Growth media treated with antibiotics or herbicides allow the selection of plants with gene edits by only permitting growth of plants producing an antibiotic -resistant protein or herbicide-resistant protein.
  • This selection agent can be used to indicate that one of the markers has been introduced through the transformation method.
  • the transformation and regeneration of corn has been described, for example, in Gordon-Kamm et al., “The Plant Cell 2: 603-618 (1990)”. Edited plants and multiplexed edited plants of the present invention that have haploid scions may also be cultivated like this.
  • crossing of a diploid scion’s flowers or floral organ(s) with a haploid inducer line is followed by permitting seed to set by the flowers or floral organ(s).
  • Methods of identifying haploid seeds are known in the art, and these methods are often used to sort the desired haploid embryos from the total progeny. These methods include the identification of known recessive phenotypes, which are always expressed in haploid tissue if present, and the employment of various methods in microscopy.
  • One aspect of the present disclosure includes a method of selecting a plant homozygous for a particular allele produced using targeted recombination including, for a plurality of plants, grafting scions onto each of the rootstocks including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scions with a haploid inducer line to produce haploid seeds, wherein the haploid seeds include the gene edit; treating the haploid seeds or plants produced from the haploid seeds with an agent to increase ploidy in the haploid seeds or the plants produced from the haploid seeds; and screening the treated seeds or plants produced from the haploid seeds or the treated seeds or plants produced from the haploid seeds or
  • One aspect of the present disclosure includes a method of selecting a plant homozygous for a particular allele produced using targeted recombination including, for a plurality of plants, grafting scions onto each of the rootstocks including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scions with a haploid inducer line to produce haploid seeds, wherein the haploid seeds include the gene edit; treating the haploid seeds or plants produced from the haploid seeds with an agent to increase ploidy in the haploid seeds or the plants produced from the haploid seeds; and screening the treated seeds or plants produced from the haploid seeds
  • One aspect of the present disclosure includes a method of selecting a plant homozygous for a particular allele produced using targeted recombination including, for a plurality of plants, grafting scions onto each of the rootstocks including nucleic acid encoding a sequence specific recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scions with a haploid inducer line to produce haploid seeds, wherein the haploid seeds include the gene edit; treating the haploid seeds or plants produced from the haploid seeds with an agent to increase ploidy in the haploid seeds or the plants produced from the haploid seeds; and screening the treated seeds or plants produced from the haploid seeds or the treated seeds or plants produced from the haploid seeds for
  • another method is provided of selecting a plant homozygous for a gene edit, including grafting a scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; growing a diploid progeny from the scion; crossing a plant grown from the diploid progeny with a haploid inducer line to produce a haploid seed, wherein the haploid seed includes the gene edit; treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed or the plant produced from the haploid seed, thereby producing a plant homozygous for the gene edit; and screening the treated seeds or
  • another method is provided of selecting a plant homozygous for a gene edit, including grafting a scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; growing a diploid progeny from the scion; crossing a plant grown from the diploid progeny with a haploid inducer line to produce a haploid seed, wherein the haploid seed includes the gene edit; treating the haploid seed or a plant produced from the haploid seed with an agent to increase ploidy in the haploid seed or the plant produced from the haploid seed, thereby producing a plant homozygous for
  • the above methods further include determining the phenotype of the haploid seed.
  • the phenotype of the haploid seed is determined by microscopy.
  • the phenotyping of the haploid seed is determined by assessing chemical content.
  • Another aspect of the present disclosure for selecting a plant homozygous for a particular allele produced using targeted recombination including, for a plurality of plants, grafting haploid scions onto each of the rootstocks including nucleic acid encoding a nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing edited haploid cells in the scion, wherein the edited haploid cell comprises a targeted recombination event; culturing the edited cells in vitro to produce edited haploid plants; treating the edited haploid plants with an agent to increase ploidy in the edited haploid plant; and screening the treated edited haploid plants for the desired ploidy and/or the particular allele produced using targeted recombination.
  • MTS meristem transport segment
  • Another aspect of the present disclosure for selecting a plant homozygous for a particular allele produced using targeted recombination including, for a plurality of plants, grafting haploid scions onto each of the rootstocks including nucleic acid encoding a nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing edited haploid cells in the scion, wherein the edited haploid cell comprises a targeted recombination event; culturing the edited cells in vitro to produce edited haploid plants; treating the edited haploid plants with an agent to increase ploidy in the edited haploid plant; and screening the treated edited haploid plants for the desired ploidy and/or the particular allele produced using targeted re
  • methods of the present disclosure include selecting the treated seeds or plants including the particular allele produced using targeted recombination.
  • the nucleic acid includes a reporter gene.
  • the reporter gene encodes a fluorescent protein.
  • the nucleic acid includes a marker gene.
  • the marker gene encodes an antibiotic -resistant protein or an herbicide-resistant protein.
  • One method for distinguishing haploid seeds from diploid seeds in a double haploid breeding program is based on the presence or absence of the visible anthocyanin marker in the embryo. This can include sorting seeds by hand, using a spectrophotometer to quantify the hue and sort out believed haploid seeds based on measurements by the spectrophotometer. Other methods include selecting haploids based on oil content of a seed or seeds, wherein haploid seeds possess lower amounts of oil, as determined by nuclear magnetic resonance (Melchinger et al. 2013, Sci Rep. 3:2129).
  • the agent to increase ploidy is applied to the haploid seed or plant produced from the haploid seed.
  • the scion is diploid.
  • Methods can involve, for example, contacting the haploid cell with nitrous oxide, anti-microtubule herbicides, or colchicine.
  • Choice of agent for chromosomal doubling can depend on many factors, including reliability or lack thereof in a given target taxon.
  • at least one plant meristem of the edited haploid plant or edited haploid plants is treated with the agent to increase ploidy.
  • the agent to increase ploidy is applied to the haploid seedling.
  • Nitrous oxide is a gaseous substance that has been demonstrated to induce chromosome doubling in barley, red clover, rice and wheat. Nitrous oxide treatment for chromosome doubling quickly and uniformly penetrates plant tissue. The plant tissue can be exposed to nitrous oxide at room temperature. Suitable amounts of nitrous oxide include about 100 kPa, 100-200 kPa, 100-300 kPa, 100-400 kPa, 100-500 kPa, 100-600 kPa, and about 100- 1400 kPa or greater amounts of N2O. Treatment time for nitrous oxide can vary, including about 1-3, 1-6, 1-12, 1-18, 1-24, 1-36, 1-48, 1-60, or about 1-72 hours. Treatment time for nitrous oxide also includes greater than 3 days, including about 4-6 days.
  • Colchicine is one of many available anti-microtubule or anti-mitotic agents for producing doubled haploid (DH) plants. Colchicine can be applied through various methods and at various concentrations. Suitable methods of applying colchicine include application of a liquid solution containing colchicine, applying beads coated in colchicine, and others. Application of colchicine may be facilitated by injection, vacuum, or root submergence.
  • Suitable concentrations of colchicine include about 10 ppm, 10-20 ppm, 10-30 ppm, 10-40 ppm, 10-50 ppm, 10-60 ppm, 10-70 ppm, 10-80 ppm, 10-90 ppm, 10-100 ppm, 10-200 ppm, 10-300 ppm, 10-400 ppm, 10-500 ppm, 10-600 ppm, 10-700 ppm, 10-800 ppm, 10-900 ppm, 10-1000 ppm, 10-2000 ppm, 10-3000 ppm, 10-4000 ppm, 10-5000 ppm, 10-6000 ppm, 10- 7000 ppm, 10-8000 ppm, 10-9000 ppm, and up to 10 to about 10,000 ppm.
  • colchicine treatments may be applied in similar ways and at comparable concentrations.
  • the agent to increase ploidy is an antimitotic agent.
  • Colchicine can be applied in various plant locations, including close to plant meristems. Accordingly, in some embodiments, at least one plant meristem of the plant produced by the haploid seed is treated with the agent to increase ploidy.
  • the agent is applied to the at least one plant meristem.
  • the antimitotic agent is colchicine.
  • the antimitotic agent is oryzalin or trifluralin.
  • the antimitotic agent is dissolved in dimethyl sulfoxide.
  • the method includes grafting a scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the gene edit; and treating the haploid seed with an agent to increase ploidy in the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination
  • the haploid seed is treated with an agent to increase ploidy in the haploid seed.
  • treating the haploid seed includes bringing the seed into contact with a composition containing the agent to increase ploidy. In some embodiments, treating the haploid seed includes treating it with a composition containing the agent to increase ploidy, with infiltrative assistance provided by injection or vacuum.
  • the method includes grafting a scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific binding protein fused to a recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the gene edit; and treating the haploid seed with an agent to increase ploidy in the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination
  • the haploid seed is treated with an agent to increase ploidy in the haploid seed.
  • treating the haploid seed includes bringing the seed into contact with a composition containing the agent to increase ploidy. In some embodiments, treating the haploid seed includes treating it with a composition containing the agent to increase ploidy, with infiltrative assistance provided by injection or vacuum.
  • the method includes grafting a scion onto a rootstock including nucleic acid encoding a sequence specific recombinase fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific recombinase cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the gene edit; and treating the haploid seed with an agent to increase ploidy in the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination
  • the haploid seed is treated with an agent to increase ploidy in the haploid seed.
  • treating the haploid seed includes bringing the seed into contact with a composition containing the agent to increase ploidy. In some embodiments, treating the haploid seed includes treating it with a composition containing the agent to increase ploidy, with infiltrative assistance provided by injection or vacuum.
  • the method includes grafting a scion onto a rootstock including nucleic acid encoding a sequence specific endonuclease fused to nucleic acid encoding a meristem transport segment (MTS), wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby producing a particular allele produced using targeted recombination in the scion; crossing the scion with a haploid inducer line to produce a haploid seed, wherein the haploid seed comprises the gene edit; and treating a plant produced from the haploid seed with an agent to increase ploidy in the plant produced from the haploid seed, thereby producing a plant homozygous for the particular allele produced using targeted recombination
  • the method includes grafting a scion onto a rootstock including nucleic acid encoding a sequence specific binding protein fused to a recombinas
  • treating the plant produced from the haploid seed includes bringing the plant into contact with a composition containing the agent to increase ploidy.
  • treating the plant produced from the haploid seed includes treating it with a composition containing the agent to increase ploidy, with infiltrative assistance provided by injection, vacuum, or root submergence.
  • At least one edited plant is produced through the methods.
  • Example 1 Transgenic expression of mobile genome editing reagents in root stocks
  • a CRISPR-Cas nuclease is codon-optimized for expression in soybean. Additional features to further increase nuclease activity include disrupting the protein coding sequence with multiple introns (Griitzner et al. Plant Commun. 2021, 2: 100135), adding a transcriptional enhancer in the T-DNA of the agrobacterium binary vector (Nuccio et al. Recent Adv. Gene. Expr. Enabling Technol. Crop Plants.
  • a meristem transport segment like the 102 bp Arabidopsis FT element (Ei et al. J Virol 2009, 83: 3540-3548; Jackson and Hong Front Plant Sci 2012, 3: 127), is fused to the 3’-UTR just after the translation stop codon and before the transcriptional terminator sequence.
  • meristem transport segments There are a variety of meristem transport segments to choose from including those based on tRNA sequence (Zhang et al.
  • a meristem transport segment like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Li et al. Sci Rep 2011, 1: 73) is fused to the 5’- or 3’-terminus of the companion guide RNA or guide RNA array to the CRISPR Cas nuclease and expressed from a suitable RNA polymerase III promoter (Hassan et al. Trends Plant Sci 2021, 26: 1133- 1152). This construct is incorporated into the same T-DNA vector that includes the gene encoding the MTS-tagged CRISPR-Cas nucleic acid.
  • the guide RNA or guide RNA array DNA sequence can be expressed from an RNA polymerase II promoter if it is flanked by a hammerhead ribozyme at the 5 ’-terminus and an HDV ribozyme at the 3 ’-terminus (Gao and Zhao J Integr Plant Biol 2014, 56: 343-349).
  • the meristem transport segment must be situated between the two ribozymes.
  • the T-DNA can also include a reporter gene such as a fluorescent protein fused to a meristem transport segment, like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Li et al. Sci Rep 2011, 1: 73) to enable tracking of meristem transport segment function in planta.
  • a reporter gene such as a fluorescent protein fused to a meristem transport segment, like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Li et al. Sci Rep 2011, 1: 73) to enable tracking of meristem transport segment function in planta.
  • a guide RNA targeting a non-essential or harmless sequence in the rootstock genome may also be included to assess CRISPR system function and aid in the selection of suitable MTS-tagged CRISPR Cas Editor plant lines.
  • Guide RNA(s) whose action might produce a harmless but
  • the MTS-tagged CRISPR system is transformed into a suitable line-and transformants are selected based on the presence of the T-DNA, fluorescent protein activity and/or CRISPR system activity.
  • a transgenic plant with a transgene that expresses a CRISPR-Cas nuclease is termed an “Editor”.
  • a transgenic plant with a transgene that expresses a mobile CRISPR-Cas nuclease is termed an “MTS-tagged CRISPR Cas Editor”.
  • the regenerates are recovered and grown to maturity to collect seed. Progeny from ideal regenerants are tested for T-DNA heritability and transgene stability. These lines are propagated as needed.
  • the seed for both the MTS-tagged CRISPR Cas Editor line and the target line(s) are germinated on germination paper or by planting in soil. About 5-7 days later the shoots of the target line(s) are grafted to the roots of the MTS-tagged CRISPR Cas Editor line(s) using standard procedures developed for soybean (Bezdicek et al. Agron J 1972, 64: 558-558), monocots like com and wheat (Reeves et al. Nature 2022, 602: 280-286), or the species of interest (Warschefsky et al. Trends Plant Sci 2016, 21: 418-437).
  • the grafted shoot is then monitored for evidence of fluorescence if a mobile reporter is present in the MTS-tagged CRISPR Cas Editor line, for phenotypic readout, and/or for the presence of the intended edits in new growth of each grafted target plant.
  • Grafted target scions with the intended edits are self-pollinated or crossed to a suitable parent and grown to maturity. The harvested seed are evaluated for inheritance of the intended edits.
  • This method enables editing of any germplasm that is graft compatible with the MTS- tagged CRISPR Cas Editor line regardless of its transformability. Edited germplasm produced this way will not inherit the transgenes used to produce the Cas nuclease or the guide RNA of the CRISPR Cas system.
  • An additional benefit is that edits can be rapidly propagated into elite commercial lines simultaneously and in a single generation, greatly reducing the time required to produce marketable material.
  • a T-DNA containing an MTS-tagged CRISPR-Cas nuclease and at least one guide RNA as described in Example 1 is transformed directly into Agrobacterium rhizogenes, which is used to infect a rootstock plant to produce hairy roots (Hao et al. Curr Biochem Eng 2021, 7: 31-37; Song et al. Curr Protoc 2021, 1: el95).
  • a variety of soybean cultivars are susceptible and produce transgenic hairy roots. The transgenic hairy roots produce the MTS- tagged Cas nuclease and at least one guide RNA which are transported to the shoot apical meristem to modify the stem cells that give rise to the reproductive structures.
  • the transformed plants are transferred to soil and grown to maturity.
  • the shoot is monitored for evidence of fluorescence, if a mobile reporter is present in the transformed T- DNA, for phenotypic readout, and/or for the presence of the intended edits in new growth of each transgenic plant.
  • Plants with the intended edits are self-pollinated or crossed to a suitable parent and grown to maturity.
  • the harvested seed are evaluated for inheritance of the intended edits.
  • Example 3 Transgenic expression of a Cas using a constitutive promoter combined with delivery of MTS-tagged guide RNAs
  • Multiple heritable edits can be introduced into an Editor line constitutively expressing a CRISPR Cas nuclease.
  • a T-DNA containing a CRISPR-Cas nuclease is designed as in Example 1, but utilizing promoters that are highly active in most plant tissues (Binet et al. Plant Mol Biol 1991, 17: 395-407; Christensen and Quail Transgenic Res 1996, 5: 213-218; Hernandez- Garcia et al. Plant Cell Rep 2009, 28: 837-849; Amack and Antunes Curr Plant Biol 2020, 24: 100179).
  • the T-DNA can also include a reporter gene such as a fluorescent protein (Schnitzler et al. Mar Biotechnol 2008, 10: 328-342) to enable assessment of T-DNA function in planta.
  • a reporter gene such as a fluorescent protein (Schnitzler et al. Mar Biotechnol 2008, 10: 328-342) to enable assessment of T-DNA function in planta.
  • a guide RNA targeting a non-essential or harmless sequence in the Editor plant genome may also be included to assess CRISPR system function and aid in the selection of suitable Editor plant lines.
  • Guide RNA(s) whose action might produce a harmless but visible signal in target gene lines, such as an obvious trichome phenotype (Wang et al. Plant Biotechnol J 2019, 17: 1706-1722) to enable assessment of CRISPR system function in target plants can also be used.
  • MTS-tagged guide RNAs or guide RNA arrays are produced using in vitro transcription (Huang and Yu Curr Protoc Mol Biol 2013, 102: 4.15.1-4.15.14) for application to Editor lines.
  • a meristem transport segment like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Li et al. Sci Rep 2011, 1: 73) is fused to the 5’- or 3’- terminus of the companion guide RNA or guide RNA array to the CRISPR Cas nuclease and expressed from a suitable RNA polymerase promoter suitable for runoff in vitro transcription, like the T7, T3 or Sp6 promoter.
  • the guide RNA or guide RNA array DNA sequence can be flanked by a hammerhead ribozyme at the 5 ’-terminus and an HDV ribozyme at the 3’-terminus (Gao and Zhao J Integr Plant Biol 2014, 56: 343-349) to produce a precisely terminated product.
  • the meristem transport segment must be situated between the two ribozyme cleavage sites.
  • the guide RNA can be modified as needed to enhance mobility (Maizel et al. Curr Opin Plant Biol 2020, 57: 52-60), stability (Filippova et al.
  • RNAs produced in vitro can be combined with RNase inhibitors and/or methylated with a m 5 C methyltransferase to reduce degradation prior to application.
  • Example 4 Application of the gRNA by RNA spray
  • Seed representing suitable Editor lines that constitutively express a CRISPR Cas nuclease are germinated in axenic culture or in soil and grown to the first trifoliate stage. Then one of several RNA spray methods (Rank and Koch Front Plant Sci 2021, 12: 755203; Dalakouras et al. Front Plant Sci 2016, 7: 1327) is used to introduce the MTS-tagged guide RNA(s) to the plant. These include formulations consisting of carbon nanodots (Doyle et al. BioRxiv, 2019: 805036), therapeutic nanoparticles (Karny et al.
  • a formulation consisting of about 50 pM of each MTS-tagged guide RNA or guide RNA array is prepared and sprayed onto the Editor line. The spray volume should be sufficient to visibly wet the leaf surface. Plants are monitored for guide RNA uptake and mobility using a fluorescent label or a phenotypic readout in new growth post application.
  • New growth is assayed for the presence of the intended edits using any acceptable method including T7E1/TIDE and/or amplicon sequence analysis (Bernabe-Orts et al. Plant Biotechnol J 2019, 17: 1971-1984; Fee et al. Plant Biotechnol J 2019, 17: 362-372).
  • the MTS-tagged guide RNA treatment can be repeated as needed to produce the desired result.
  • Plants with the intended edits are grown to maturity and the progeny are evaluated for inheritance of the intended edits. Progeny that contain edits are retained. These progeny will not inherit the editing transgenes.
  • Seeds representing suitable Editor lines that constitutively express a CRISPR Cas nuclease are germinated in axenic culture or in soil and grown to the first trifoliate stage.
  • An approximately 50 pM solution of each MTS-tagged guide RNA or guide RNA array is prepared in nuclease-free water or phosphate buffer and 1-5 pF is injected in the stem of each Editor seedling, with the injection point being 3-5 cm below the top of the plant. Plants are monitored for guide RNA uptake and mobility using a fluorescent label or a phenotypic readout in new growth, toward the plant apex, post application.
  • New growth is assayed for the presence of the intended edits using any acceptable method including T7E1/TIDE and/or amplicon sequence analysis (Bemabe-Orts et al. Plant Biotechnol J 2019, 17: 1971-1984; Fee et al. Plant Biotechnol J 2019, 17: 362-372).
  • the MTS-tagged guide RNA treatment can be repeated as needed to produce the desired result.
  • Plants with the intended edits are grown to maturity and the progeny are evaluated for inheritance of the intended edits. Progeny that contain edits are retained. Progeny that inherit the edit but not the transgenes are selected.
  • Seeds representing suitable Editor lines that constitutively express a CRISPR Cas nuclease are germinated in axenic culture or in soil and grown to the first trifoliate stage.
  • An approximately 50 pM solution of each MTS-tagged guide RNA or guide RNA array is prepared in nuclease-free water or phosphate buffer, with or without a wetting agent such as Silwet-77.
  • the surface of the first expanded leaf is gently wounded using an abrasive agent such as glass beads or 400 grit sandpaper and 1-5 pL of the guide RNA solution is applied to the wound site.
  • Plants are monitored for guide RNA uptake and mobility using a fluorescent label or a phenotypic readout in new growth, toward the plant apex, post application. New growth is assayed for the presence of the intended edits using any acceptable method including T7E1/TIDE and/or amplicon sequence analysis (Bemabe-Orts et al. Plant Biotechnol J 2019, 17: 1971-1984; Lee et al. Plant Biotechnol J 2019, 17: 362-372). The MTS-tagged guide RNA treatment can be repeated as needed to produce the desired result. Plants with the intended edits are grown to maturity and the progeny are evaluated for inheritance of the intended edits. Progeny that contain edits are retained. Progeny that inherit the edit but not the transgenes are selected.
  • Seeds representing suitable Editor lines that constitutively express a CRISPR Cas nuclease are germinated on germination paper for 1-3 days.
  • An approximately 50 pM solution of each MTS-tagged guide RNA or guide RNA array is prepared in nuclease-free water or phosphate buffer, with or without a wetting agent like Silwet-77.
  • Each seedling is placed in the MTS-tagged guide RNA solution and incubated overnight in a humid chamber. The treated seedlings are then transferred to soil. Plants are monitored for guide RNA uptake and mobility using a fluorescent label or a phenotypic readout in new growth, toward the plant apex, post application.
  • New growth is assayed for the presence of the intended edits using any acceptable method including T7E1/TIDE and/or amplicon sequence analysis (Bemabe-Orts et al. Plant Biotechnol J 2019, 17: 1971-1984; Lee et al. Plant Biotechnol J 2019, 17: 362-372).
  • the MTS-tagged guide RNA treatment can be repeated as needed to produce the desired result.
  • Plants with the intended edits are grown to maturity and the progeny are evaluated for inheritance of the intended edits. Progeny that contain edits are retained. Progeny that inherit the edit but not the transgenes are selected.
  • Example 8 Transgenic expression of MTS-tagged Cas nuclease in rootstock, enabling editing in elite germplasm by grafting target shoots to transgenic root stock.
  • Multiple heritable edits can be introduced into an Editor rootstock line constitutively expressing an MTS-tagged CRISPR Cas nuclease.
  • a T-DNA containing a CRISPR-Cas nuclease is designed and produced as in Example 3, but with an MTS-tagged Cas nuclease.
  • An MTS like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Jackson and Hong Front Plant Sci 2012, 3: 127), is fused to the 3’-UTR just after the translation stop codon and before the transcriptional terminator sequence.
  • meristem transport segments There are a variety of meristem transport segments to choose from including those based on tRNA sequence (Zhang et al. Plant Cell 2016, 28: 1237-1249) or derived from genes that produce phloem mobile RNAs (Thieme et al. Nat Plants 2015, 1: 1-9).
  • the T-DNA can also include a reporter gene such as a fluorescent protein (Schnitzler et al. Mar Biotechnol 2008, 10: 328-342) fused to an MTS , like the 102 bp Arabidopsis FT element (Li et al. J Virol 2009, 83: 3540-3548; Li et al. Sci Rep 2011, 1: 73) to enable tracking of meristem transport segment function in planta.
  • a guide RNA targeting a non- essential or harmless sequence in the editor plant genome may also be included to assess CRISPR system function and aid in the selection of suitable MTS-tagged CRISPR Cas Editor plant lines.
  • Guide RNA(s) whose action might produce a harmless but visible signal in target gene lines, such as an obvious trichome phenotype (Wang et al. Plant Biotechnol J 2019, 17: 1706-1722), can also be linked to the MTS to enable assessment of CRISPR system function in target plants.
  • the MTS-tagged CRISPR system is transformed into a suitable line and transformants are selected based on the presence of the T-DNA, fluorescent protein activity, and/or CRISPR system activity.
  • the ideal MTS-tagged CRISPR Cas Editor line has a high fluorescent protein signal and a highly active CRISPR system based on analysis of the harmless/non-essential target site using any suitable tool including T7E1/TIDE and/or amplicon sequencing (Bemabe-Orts et al. Plant Biotechnol J 2019, 17: 1971-1984; Lee et al. Plant Biotechnol J 2019, 17: 362-372).
  • T-DNA copy number is a secondary criterium to robust, stable CRISPR system activity in healthy regenerants. The regenerates are recovered and grown to maturity to collect seed. Progeny from ideal regenerants are tested for T-DNA heritability and transgene stability. These lines are propagated as needed.
  • the seed for both the MTS-tagged CRISPR Cas Editor line and the target line(s) are germinated on germination paper or by planting in soil. About 5-7 days later the shoots of target line(s) are grafted to the roots of the MTS-tagged CRISPR Cas Editor line(s) using standard procedures developed for soybean (Bezdicek et al. Agron J 1972, 64: 558-558), monocots like com and wheat (Reeves et al. Nature 2022, 602: 280-286), or the species of interest (Warschefsky et al. Trends Plant Sci 2016, 21: 418-437). The grafted shoot is then monitored for evidence of fluorescence (if a mobile reporter is present in the MTS-tagged CRISPR Cas Editor line), phenotypic readout and/or the presence of the intended edits in new growth of each grafted plant.
  • MTS-tagged guide RNAs or guide RNA arrays are produced using in vitro transcription (Huang and Yu Curr Protoc Mol Biol 2013, 102: 4.15.1-4.15.14) for application to the MTS-tagged CRISPR Cas nuclease Editor lines.
  • a meristem transport segment, like the 102 bp Arabidopsis FT element Li et al. J Virol 2009, 83: 3540-3548; Li et al.
  • RNA polymerase promoter suitable for runoff in vitro transcription like the T7, T3 or Sp6 promoter.
  • the guide RNA or guide RNA array DNA sequence can be flanked by a hammerhead ribozyme at the 5 ’-terminus and an HDV ribozyme at the 3’-terminus (Gao and Zhao J Integr Plant Biol 2014, 56: 343-349) to produce a precisely terminated product.
  • the meristem transport segment must be situated between the two ribozyme cleavage sites.
  • the guide RNA can be modified as needed to enhance mobility (Maizel et al. Curr Opin Plant Biol 2020, 57: 52-60), stability (Filippova et al. Biochimie 2019, 167: 49-60; Rozners J Am Chem Soc 2022, 144: 12584-12594) and to enable tracking (Awwad et al. MethodsX 2020, 7: 101148) when applied to plants.
  • Suitable grafted MTS-tagged CRISPR Cas nuclease Editor lines are grown to the first trifoliate stage.
  • the method of any of Examples 4-7 is used to introduce MTS-tagged gRNA(s) to the plant.
  • Example 9 Generation of double stranded breaks at specific genome locations for targeted recombination in Fl scions grafted onto rootstocks expressing a mobile Cas
  • This Example demonstrates a targeted recombination approach for precision breeding, whereby double stranded DNA breaks can be generated at specific genome locations to trigger targeted recombination in Fl scions grafted onto rootstocks expressing a mobile Cas.
  • Fl plants resulting from breeding crosses serve as scions and are grafted onto rootstocks expressing a mobile mRNA encoding a Cas nuclease.
  • a multi-gRNA array is designed for multiplex editing to generate double- strand breaks at specific genome locations where targeted recombination events are desired.
  • the multi-gRNA array for multiplex editing is introduced via viral infection on the leaves of the scion.
  • haploid seeds are induced for double haploid production.
  • the DH lines are genotyped and characterized for recombination profiling. The recombination events will be identified in each of the DH lines. The hypothesis is that the targeted genome locations for recombination will result in more frequent recombination at the targeted location than expected compared to non-targeted meiosis process.
  • the DH lines exhibiting the desired targeted recombination are selected for breeding.
  • the F2 plants are selected via molecular characterization for downstream breeding selection and evaluation.
  • Table 3 Progeny production and selection with targeted recombination for soybean.
  • Example 10 Directed chromosomal crossover in a hybrid scion for inbred development by haploid induction and chromosome doubling
  • An Editor rootstock line constitutively expressing an MTS-tagged CRISPR Cas nuclease described in Example 8 can be grafted to a maize Fl hybrid plant (target line for editing) to trigger directed recombination events to stack beneficial alleles from the parents of the hybrid lines into a single progeny.
  • the seed for both the MTS-tagged CRISPR Cas Editor line and the target line(s) are germinated on germination paper or by planting in soil. About 5-7 days later, the shoots of target line(s) are grafted to the roots of the MTS-tagged CRISPR Cas Editor line(s) using standard procedures developed for soybean (Bezdicek et al. Agron J 1972, 64: 558-558), monocots like corn and wheat (Reeves et al. Nature 2022, 602: 280-286), or the species of interest (Warschefsky et al. Trends Plant Sci 2016, 21: 418-437).
  • Examples 4-7 The methods of any of Examples 4-7 are used to introduce MTS-tagged gRNA(s) to the plant to generate the double stranded DNA breaks.
  • the female gametes are hybridized with sperm cells in the pollen of a haploid induction line to produce haploid seeds with directed chromosomal crossovers enabled by the targeted double stranded DNA breaks.
  • the haploid seeds with directed chromosomal crossovers are subject to chromosome doubling through colchicine treatment to produce favorable inbred lines for product development.
  • Example 11 Directed chromosomal crossover in a hybrid scion for variety development by inbreeding
  • An Editor rootstock line constitutively expressing an MTS-tagged CRISPR Cas nuclease described in Example 8 can be grafted to an Fl hybrid plant (target line for editing) to trigger directed recombination events to stack beneficial alleles from the parents of the hybrid lines into a single progeny.
  • the seed for both the MTS-tagged CRISPR Cas Editor line and the target line(s) are germinated on germination paper or by planting in soil. About 5-7 days later, the shoots of target line(s) are grafted to the roots of the MTS-tagged CRISPR Cas Editor line(s) using standard procedures developed for soybean (Bezdicek et al. Agron J 1972, 64: 558-558), monocots like corn and wheat (Reeves et al. Nature 2022, 602: 280-286), or the species of interest (Warschefsky et al. Trends Plant Sci 2016, 21: 418-437).
  • Examples 4-7 The methods of any of Examples 4-7 are used to introduce MTS-tagged gRNA(s) to the plant to generate the double stranded DNA breaks. Both male and female gametes are hybridized through pollination (selfing or cross-pollination from the same or different scions, respectively, depending on breeding design) to produce seeds with directed chromosomal crossovers enabled by the targeted double stranded DNA breaks. The progeny seeds with directed chromosomal crossovers are subject to product development or recurrent recombinant selection through repeated directed chromosomal crossover creations and selections.
  • Embodiment 1 A method of inducing targeted recombination in a scion, comprising: a) providing a rootstock expressing a sequence specific endonuclease, wherein a nucleic acid encoding the sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific endonuclease cleaves a target sequence in a genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • Embodiment 2 The method of embodiment 1, wherein mRNA encoding the sequence specific endonuclease is fused to an MTS.
  • Embodiment 3 The method of embodiment 1 or embodiment 2, wherein the sequence specific endonuclease is a zinc finger nuclease (ZFN), a TALEN or a Cas protein.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • Embodiment 4 The method of embodiment 3, wherein the sequence specific endonuclease is a Cas protein.
  • Embodiment 5 A method of inducing targeted recombination in a scion, comprising: a) providing a rootstock expressing a sequence specific binding protein, wherein the sequence specific binding protein is fused to a recombinase, and wherein a nucleic acid encoding the sequence specific binding protein fused to the recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific binding protein binds to a target sequence in a genomic locus of the scion; wherein the recombinase induces cleavage and/or recombination in the genomic locus of the scion, thereby inducing targeted recombination in the scion.
  • Embodiment 6 The method of embodiment 5, wherein mRNA encoding the sequence specific binding protein fused to a recombinas
  • Embodiment 7 The method of embodiment 5, wherein the sequence specific binding protein is selected from the group consisting of: inactive zinc finger nuclease (ZFN) proteins, inactive TALEN proteins, catalytically inactive Cas proteins, and transcription factors.
  • ZFN zinc finger nuclease
  • TALEN inactive TALEN proteins
  • Cas proteins catalytically inactive Cas proteins
  • Embodiment 8 The method of embodiment 5, wherein the sequence specific binding protein comprises one or more domains of a catalytically inactive Cas protein.
  • Embodiment 9 The method of any one of embodiments 1-8, further comprising delivering a guide nucleic acid that is complementary to the target sequence in the scion.
  • Embodiment 10 A method of inducing targeted recombination in a scion, comprising: a) providing a rootstock expressing a sequence specific recombinase, wherein a nucleic acid encoding the sequence specific recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and b) grafting the scion onto the rootstock; wherein the sequence specific recombinase of the rootstock binds to a recombinase site in the genome of the scion; wherein the recombinase induces cleavage and/or recombination in the genome of the scion, thereby inducing targeted recombination in the scion.
  • MTS meristem transport segment
  • Embodiment 11 The method of embodiment 10, wherein mRNA encoding the sequence specific recombinase is fused to an MTS.
  • Embodiment 12 The method of any one of embodiments 1, 5, and 10, wherein the scion is not of a uniform or inbred line.
  • Embodiment 13 The method of embodiment 10, wherein the sequence specific recombinase comprises one or more domains of a serine recombinase and/or a tyrosine recombinase.
  • Embodiment 14 The method of embodiment 10, wherein the sequence specific recombinase binds specifically to recombination sites within the genome of the scion.
  • Embodiment 15 The method of any one of embodiments 1-14, comprising inducing DNA cleavage and/or recombination in a plurality of plants at different genomic loci, wherein the genetic diversity in the plurality of plants is increased following the cleavage and/or recombination compared to the genetic diversity in the plurality of plants prior to the cleavage and/or recombination.
  • Embodiment 16 The method of any one of embodiments 1-15, wherein the cleavage is a double- stranded break (DSB), a single-stranded break, a transposase-mediated DNA exchange reaction, or a recombinase-mediated DNA exchange reaction.
  • DSB double- stranded break
  • Embodiment 17 The method of any one of embodiments 1-16, wherein the induced targeted recombination does not comprise recombination with exogenously provided DNA.
  • Embodiment 18 The method of any one of embodiments 9 or 15-17, comprising delivering a plurality of different guide nucleic acids, wherein the plurality of different guide nucleic acids is complementary to more than one target sequence in one or more genetic loci of the scion.
  • Embodiment 19 The method of embodiment 18, wherein the target sequences are on different loci on the same chromosome, on different loci on homologous chromosomes, and/or on the same locus on homologous chromosomes.
  • Embodiment 20 The method of embodiment 18, wherein the target sequences are on the same locus on homologous chromosomes.
  • Embodiment 21 The method of any one of embodiments 18-20, wherein the genomic loci are at least 1 centimorgan (cM) apart from each other on the genome of the scion.
  • cM centimorgan
  • Embodiment 22 The method of any one of embodiments 1-21, wherein the target sequence is within an intergenic region.
  • Embodiment 23 The method of any one of embodiments 1-21, wherein at least one target sequence is within a regulatory element, an intron, or an exon.
  • Embodiment 24 The method of embodiment 23, wherein the regulatory element is in a promotor.
  • Embodiment 25 The method of any of embodiments 1-24, wherein the target sequence is within 50 bp of the 5’ or 3’ end of a gene of interest, wherein the target sequence is within 50 bp of a polymorphism of interest, and/or wherein the target sequence is within a genomic region that has sufficient surrounding 5’ and 3’ identity to allow for recombination.
  • Embodiment 26 The method of any of embodiments 9 and 13-25, wherein the guide nucleic acid is a gRNA.
  • Embodiment 27 The method of any one of embodiments 1-26, wherein the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase, is constitutively expressed in the rootstock.
  • Embodiment 28 The method of any one of embodiments 1-27, wherein RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported from the rootstock to the scion by the plant vascular system.
  • Embodiment 29 The method of any one of embodiments 1-28, wherein RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported to the scion through the xylem or the phloem.
  • Embodiment 30 The method of any one of embodiments 1-29, wherein RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is transported to the meristem and/or a somatic region of the scion.
  • Embodiment 31 The method of any one of embodiments 1-30, wherein RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is translated in the scion.
  • Embodiment 32 The method of any one of embodiments 1-31, wherein RNA encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is translated in a meristem cell of the scion and/or a somatic cell of the scion.
  • Embodiment 33 The method of any one of embodiments 1-32, wherein targeted recombination is induced in a meristem cell and/or a scion cell.
  • Embodiment 34 The method of any one of embodiments 1-33, wherein the scion and the rootstock are different plant species.
  • Embodiment 35 The method of any one of embodiments 1-33, wherein the scion and the rootstock are the same plant species.
  • Embodiment 36 The method of any one of embodiments 1-35, wherein the scion and/or rootstock is a dicot.
  • Embodiment 37 The method of any one of embodiments 1-35, wherein the scion and/or rootstock is a monocot.
  • Embodiment 38 The method of any one of embodiments 9 and 15-37, wherein the guide nucleic acid is provided by providing a vector that encodes the guide nucleic acid, optionally wherein the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase and the nucleic acid encoding the guide nucleic acid are provided in different vectors.
  • Embodiment 39 The method of any one of embodiments 18-38, wherein the plurality of different guide nucleic acids are provided in a single vector that encodes each of the guide nucleic acids.
  • Embodiment 40 The method of any one of embodiments 18-38, wherein the plurality of different guide nucleic acids are provided in multiple different vectors, wherein each vector encodes one or more different guide nucleic acids.
  • Embodiment 41 The method of any one of embodiments 9 and 15-40, wherein the guide nucleic acid or plurality of different guide nucleic acids is delivered to the scion in a viral vector or a T-DNA vector comprising nucleic acid encoding the guide nucleic acid.
  • Embodiment 42 The method of any one of embodiments 9 and 15-41, the wherein delivery of the guide nucleic acid or plurality of different guide nucleic acids comprises spraying a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • Embodiment 43 The method of any one of embodiments 9 and 15-42, the wherein delivery of the guide nucleic acid or plurality of different guide nucleic acids comprises incubating the scion with a composition comprising the guide nucleic acid.
  • Embodiment 44 The method of any one of embodiments 9 and 15-43, the wherein delivery of the guide nucleic acid comprises transformation of the scion by Agrobacterium rhizogenes or Agrobacterium tumefaciens.
  • Embodiment 45 The method of any one of embodiments 1-44, wherein the MTS is a Flowering Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.
  • FT Flowering Locus T
  • TLS tRNA like sequence
  • MTC meristem transport component
  • RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.
  • Embodiment 46 The method of embodiment 45, wherein the FT-derived sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • Embodiment 47 The method of embodiments 45, wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.
  • Embodiment 48 The method of any one of embodiments 9 and 15-47, wherein the guide nucleic acid or plurality of different guide nucleic acids are fused to a meristem transport segment (MTS).
  • MTS meristem transport segment
  • Embodiment 49 The method of any one of embodiments 1-48, wherein the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase.
  • Embodiment 50 The method of any one of embodiments 1-48, wherein the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase.
  • Embodiment 51 The method of any one of embodiments 1-50, wherein the nucleic acid encoding the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is operably linked to a promoter.
  • Embodiment 52 The method of embodiment 51 wherein the promoter is active in roots and/or phloem companion cells.
  • Embodiment 53 The method of embodiment 52, wherein the promoter is the promoter of a gene selected from the group consisting of Arabidopsis WRKY6, chickpea WRKY31, carrot MYB113, com GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof.
  • Embodiment 54 The method of embodiment 52, wherein the promoter is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FORI gene, a rice tungro bacilliform virus promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle vims promoter, a wheat dwarf vims promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloemspecific isoform of plasmamembrane H+-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter.
  • FT Flowering Locus T
  • Fabaceaen FORI Fabaceaen FORI gene
  • rice tungro bacilliform virus promoter an RmlC-like cupins superfamily protein promoter
  • Embodiment 55 The method of embodiment 52, wherein the promoter is a constitutive promoter.
  • Embodiment 56 The method of embodiment 55, wherein the constitutive promoter is a ubiquitin promoter.
  • Embodiment 57 The method of any one of embodiments 1-56, wherein the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in dicots.
  • Embodiment 58 The method of any one of embodiments 1-56, wherein the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in monocots.
  • Embodiment 59 The method of any one of embodiments 1-56, wherein the sequence specific endonuclease, sequence specific binding protein, or sequence specific recombinase is codon-optimized for expression in com, soy, or wheat.
  • Embodiment 60 The method of any one of embodiments 9 and 15-59, wherein nucleic acid(s) encoding the guide nucleic acid(s) is operably linked to a promoter.
  • Embodiment 61 The method of embodiment 60, wherein the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.
  • Embodiment 62 The method of embodiment 60, wherein the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the rootstock.
  • Embodiment 63 The method of embodiment 60, wherein the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the scion.
  • Embodiment 64 The method of any one of embodiments 9 and 15-63, wherein nucleic acid(s) encoding the guide nucleic acid(s) and the MTS is/are located between two ribozyme sequences.
  • Embodiment 65 The method of embodiment 64, wherein each of the ribozyme sequences is independently selected from the group consisting of a hammerhead ribozyme sequence, a HDV ribozyme sequence, a Csy4 sequence, and a tRNA processing enzyme sequence.
  • Embodiment 66 The method of any one of embodiments 18-65, wherein the method comprises delivering two or more, three or more, four or more, or five or more different guide nucleic acids.
  • Embodiment 67 The method of embodiment 66, wherein the two or more, three or more, four or more, or five or more different guide nucleic acids are each joined to an MTS.
  • Embodiment 68 The method of any one of embodiments 3-4, 7-9, and 15-67, wherein the Cas is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j.
  • Embodiment 69 The method of any one of embodiments 3-4, 7-9, and 15-67, wherein the Cas is a Cas nickase.
  • Embodiment 70 The method of embodiment 69, wherein the Cas nickase is a Cas9 nickase or a Cas 12 nickase.
  • Embodiment 71 The method of any one of embodiments 68-70, wherein the Cas nickase comprises mutation in one or more nuclease active sites.
  • Embodiment 72 The method of any one of embodiments 1-71, wherein the rootstock and/or scion further comprises a nucleic acid encoding a detectable marker fused to a nucleic acid encoding a MTS.
  • Embodiment 73 The method of any one of embodiments 9 and 15-72, wherein the guide nucleic acid comprises a 5-methycytosine group.
  • Embodiment 74 The method of any one of embodiments 9 and 15-73, wherein nucleic acid encoding the guide nucleic acid and the MTS further comprises a hammerhead ribozyme sequence 5’ to the nucleic acid encoding the guide nucleic acid and the MTS, and a HDV ribozyme 3’ to the nucleic acid encoding the guide nucleic acid and the MTS.
  • Embodiment 75 The method of any one of embodiments 9 and 15-74, wherein the nucleic acid encoding the guide nucleic acid and the MTS further comprises a terminator.
  • Embodiment 76 The method of embodiment 75, wherein the terminator is a U6 terminator.
  • Embodiment 77 The method of any one of embodiments 1-76, further comprising selecting a scion comprising a somatic cell and/or a meristem cell comprising the recombined genomic locus.
  • Embodiment 78 The method of any one of embodiment 77, further comprising producing a seed, protoplast, or cell culture from the somatic cell and/or the meristem cell of the selected scion, wherein the seed, protoplast, or cell culture comprises the recombined genomic locus.
  • Embodiment 79 The method of embodiment 78, further comprising regenerating a plant from the seed, protoplast, or cell culture.
  • Embodiment 80 The method of any one of embodiments 1-79, further comprising retrieving a progeny of the scion, wherein the genome of the progeny comprises the recombined genomic locus.
  • Embodiment 81 The method of any one of embodiments 9 and 15-80, wherein the guide nucleic acid further comprises: (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide nucleic acid; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide nucleic acid; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar.
  • Embodiment 82 The method of embodiment 81, wherein each of the one or more modified nucleotides is independently selected from the group consisting of a 2'-O-methyl nucleotide, a 2'-O-methyl-3'-phosphorothioate nucleotide, a 2'-O-methyl-3'- phosphonoacetate nucleotide, and a 2'-O-methyl-3'-phosphonothioacetate nucleotide.
  • Embodiment 83 The method of embodiment 81, wherein the one or more modified nucleotide comprises a modified internucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group.
  • Embodiment 84 The method of any one of embodiments 43 and 45-83, wherein the composition comprising the guide nucleic acid comprises a surfactant.
  • Embodiment 85 The method of any one of embodiments 43 and 45-83, wherein the composition comprising the guide nucleic acid comprises glass beads coated with the guide nucleic acid.
  • Embodiment 86 The method of any one of embodiments 43 and 45-85, wherein delivery of the guide nucleic acid comprises rubbing a composition comprising the guide nucleic acid onto the leaves, shoot, stem, and/or meristem.
  • Embodiment 87 The method of any one of embodiments 43 and 45-86 wherein delivery of the guide nucleic acid comprises injecting a composition comprising the guide nucleic acid into the stem of the scion.
  • Embodiment 88 The method of any one of embodiments 43 and 45-87, wherein delivery of the guide nucleic acid comprises leaf infiltration of a composition comprising the guide nucleic acid into the leaf.
  • Embodiment 89 The method of embodiment 88, wherein the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.
  • Embodiment 90 The method of any one of embodiments 43 and 45-89 wherein the composition comprising the guide nucleic acid comprises a nuclease inhibitor.
  • Embodiment 91 The method of embodiment 90, wherein the nuclease inhibitor comprises an RNase inhibitor.
  • Embodiment 92 The method of any one of embodiments 9 and 15-91, wherein delivery of the guide nucleic acid comprises biolistic transformation of nucleic acid encoding the guide nucleic acid into the leaf, shoot, stem, and/or meristem.
  • Embodiment 93 The method of embodiment 92, wherein the biolistic transformation comprises transformation of circular DNA encoding the guide nucleic acid.
  • Embodiment 94 The method of any one of embodiments 18-93, wherein the plurality of different guide nucleic acids comprises two or more guide RNAs that are encoded by a single precursor RNA.
  • Embodiment 95 The method of embodiment 94, wherein the two or more guide RNAs are each flanked by a direct repeat.
  • Embodiment 96 The method of any of embodiments 1-95, further comprising: c) inducing the formation of haploid seeds; d) producing double haploid lines from said haploid seeds; and optionally e) genotyping and/or characterizing the double haploid lines for recombination profiling.
  • Embodiment 97 The method of any of embodiments 1-96, further comprising: c) producing one or more F2 lines from the scion by allowing the scion to self-pollinate; d) selecting an F2 line comprising a desired recombination; and optionally e) producing additional lines from the selected F2 line by haploid production and/or genome doubling.
  • Embodiment 98 The method of any of embodiments 1-97, wherein the scion is from an Fl plant.
  • Embodiment 99 The method of embodiment 98, wherein the recombination stacks beneficial alleles from the parents of Fl plant into a single progeny.
  • Embodiment 100 The method of any of embodiments 1-99, wherein the scion is diploid.
  • Embodiment 101 The method of any of embodiments 1-99, wherein the scion is haploid.
  • Embodiment 102 The method of any of embodiments 1-101, wherein the scion and/or rootstock is from a soy, maize, canola, alfalfa, com, oat, sorghum, sugarcane, banana or wheat plant.
  • Embodiment 103 The method of any of embodiments 1-102, wherein the scion and/or rootstock is from a soy, maize, or wheat plant.
  • Embodiment 104 The method of embodiment 80, further comprising inducing targeted recombination in a scion of the progeny by subjecting the scion of the progeny to the methods of any of embodiments 1-79 to produce one or more additional recombined loci.
  • Embodiment 105 The method of embodiment 104, further comprising retrieving one or more progeny comprising the one or more additional recombined loci.
  • Embodiment 106 The method of embodiment 105, further comprising inducing targeted recombination in a scion of the one or more progeny by subjecting the scion of the one or more progeny to the methods of any of embodiments 1-79 to produce one or more other recombined loci.
  • Embodiment 107 A plant produced by the method of any of embodiments 1-106.
  • Embodiment 108 A seed produced by the plant of embodiment 107.
  • Embodiment 109 A plant grown from the seed of embodiment 108.
  • Embodiment 110 A plant genome produced by the targeted recombination of the method of any of embodiments 1-106.
  • Embodiment 111 An isolated plant cell produced by the method of any of embodiments 1-106, wherein the plant genome is recombined at the target sequence compared to a corresponding genome that was not subjected to the method.
  • a plant comprising a scion grafted onto a rootstock, wherein the rootstock comprises: (i) a heterologous sequence specific endonuclease and/or a nucleic acid encoding a heterologous sequence specific nuclease, wherein a nucleic acid encoding the sequence specific endonuclease is fused to a nucleic acid encoding a meristem transport segment (MTS); (ii) a heterologous sequence specific binding protein and/or a nucleic acid encoding a heterologous sequence specific binding protein, wherein the sequence specific binding protein is fused to a recombinase, and wherein a nucleic acid encoding the sequence specific binding protein fused to the recombinase is fused to a nucleic acid encoding a meristem transport segment (MTS); and/or (iii) a heterologous sequence specific recombinase and/or a nucleic acid

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Abstract

Selon certains aspects, la présente invention concerne des procédés d'induction d'une recombinaison ciblée dans un greffon qui est greffé sur un porte-greffe, le porte-greffe exprimant un ARNm codant pour une endonucléase spécifique de séquence, une protéine de liaison spécifique de séquence fusionnée à une recombinase, et/ou une recombinase spécifique de séquence, l'ARNm codant pour l'endonucléase spécifique de séquence, une protéine de liaison spécifique de séquence fusionnée à une recombinase, et/ou une recombinase spécifique de séquence étant fusionnée à un segment de transport de méristème.
PCT/US2024/040615 2023-08-04 2024-08-01 Recombinaison ciblée Pending WO2025034520A2 (fr)

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